Methods for producing hyaluronan in a recombinant host cell

ABSTRACT

The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a  Bacillus  host cell under conditions suitable for production of the hyaluronic acid, wherein the  Bacillus  host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium. The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/084,230filed Apr. 11, 2011, which is a divisional of U.S. application Ser. No.12/891,548 filed Sep. 27, 2010, which is a divisional of U.S.application Ser. No. 10/326,185 filed Dec. 20, 2002, now U.S. Pat. No.7,811,806, which claims priority from U.S. Provisional Application Ser.No. 60/342,644 filed Dec. 21, 2001, which applications are fullyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for producing a hyaluronan in arecombinant host cell.

2. Description of the Related Art

The most abundant heteropolysaccharides of the body are theglycosaminoglycans. Glycosaminoglycans are unbranched carbohydratepolymers, consisting of repeating disaccharide units (only keratansulphate is branched in the core region of the carbohydrate). Thedisaccharide units generally comprise, as a first saccharide unit, oneof two modified sugars—N-acetylgalactosamine (GalNAc) orN-acetylglucosamine (GlcNAc). The second unit is usually an uronic acid,such as glucuronic acid (GlcUA) or iduronate.

Glycosaminoglycans are negatively charged molecules, and have anextended conformation that imparts high viscosity when in solution.Glycosaminoglycans are located primarily on the surface of cells or inthe extracellular matrix. Glycosaminoglycans also have lowcompressibility in solution and, as a result, are ideal as aphysiological lubricating fluid, e.g., joints. The rigidity ofglycosaminoglycans provides structural integrity to cells and providespassageways between cells, allowing for cell migration. Theglycosaminoglycans of highest physiological importance are hyaluronan,chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate, andkeratan sulfate. Most glycosaminoglycans bind covalently to aproteoglycan core protein through specific oligosaccharide structures.Hyaluronan forms large aggregates with certain proteoglycans, but is anexception as free carbohydrate chains form non-covalent complexes withproteoglycans.

Numerous roles of hyaluronan in the body have been identified (see,Laurent T. C. and Fraser J. R. E., 1992, FASEB J. 6: 2397-2404; andToole B. P., 1991, “Proteoglycans and hyaluronan in morphogenesis anddifferentiation.” In: Cell Biology of the Extracellular Matrix, pp.305-341, Hay E. D., ed., Plenum, New York). Hyaluronan is present inhyaline cartilage, synovial joint fluid, and skin tissue, both dermisand epidermis. Hyaluronan is also suspected of having a role in numerousphysiological functions, such as adhesion, development, cell motility,cancer, angiogenesis, and wound healing. Due to the unique physical andbiological properties of hyaluronan, it is employed in eye and jointsurgery and is being evaluated in other medical procedures. Products ofhyaluronan have also been developed for use in orthopaedics,rheumatology, and dermatology.

Rooster combs are a significant commercial source for hyaluronan.Microorganisms are an alternative source. U.S. Pat. No. 4,801,539discloses a fermentation method for preparing hyaluronic acid involvinga strain of Streptococcus zooepidemicus with reported yields of about3.6 g of hyaluronic acid per liter. European Patent No. EP0694616discloses fermentation processes using an improved strain ofStreptococcus zooepidemicus with reported yields of about 3.5 g ofhyaluronic acid per liter.

The microorganisms used for production of hyaluronic acid byfermentation are strains of pathogenic bacteria, foremost among thembeing several Streptococcus spp. The group A and group C streptococcisurround themselves with a nonantigenic capsule composed of hyaluronan,which is identical in composition to that found in connective tissue andjoints. Pasteurella multocida, another pathogenic encapsulatingbacteria, also surrounds its cells with hyaluronan.

Hyaluronan synthases have been described from vertebrates, bacterialpathogens, and algal viruses (DeAngelis, P. L., 1999, Cell. Mol. LifeSci. 56: 670-682). WO 99/23227 discloses a Group I hyaluronate synthasefrom Streptococcus equisimilis. WO 99/51265 and WO 00/27437 describe aGroup II hyaluronate synthase from Pasturella multocida. Ferretti et al.disclose the hyaluronan synthase operon of Streptococcus pyogenes, whichis composed of three genes, hasA, hasB, and hasC, that encodehyaluronate synthase, UDP glucose dehydrogenase, and UDP-glucosepyrophosphorylase, respectively (Proc. Natl. Acad. Sci. USA. 98,4658-4663, 2001). WO 99/51265 describes a nucleic acid segment having acoding region for a Streptococcus equisimilis hyaluronan synthase.

Bacilli are well established as host cell systems for the production ofnative and recombinant proteins. It is an object of the presentinvention to provide methods for producing a hyaluronan in a recombinantBacillus host cell.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to methods for producing a hyaluronicacid, comprising: (a) cultivating a Bacillus host cell under conditionssuitable for production of the hyaluronic acid, wherein the Bacillushost cell comprises a nucleic acid construct comprising a hyaluronansynthase encoding sequence operably linked to a promoter sequenceforeign to the hyaluronan synthase encoding sequence; and (b) recoveringthe hyaluronic acid from the cultivation medium.

In preferred embodiments, the nucleic acid construct further comprisesone or more genes encoding enzymes in the biosynthesis of a precursorsugar of the hyaluronic acid or the Bacillus host cell further comprisesone or more second nucleic acid constructs comprising one or more genesencoding enzymes in the biosynthesis of the precursor sugar.

In another preferred embodiment, the one or more genes encoding aprecursor sugar are under the control of the same or a differentpromoter(s) as the hyaluronan synthase encoding sequence.

The present invention also relates to Bacillus host cells comprising anucleic acid construct comprising a hyaluronan synthase encodingsequence operably linked to a promoter sequence foreign to thehyaluronan synthase encoding sequence, and to such nucleic acidconstructs.

The present invention also relates to an isolated nucleic acid sequenceencoding a hyaluronan synthase operon comprising a hyaluronan synthasegene or a portion thereof and a UDP-glucose 6-dehydrogenase gene, andoptionally one or more genes selected from the group consisting of aUDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosaminepyrophosphorylase gene, and glucose-6-phosphate isomerase gene.

The present invention also relates to isolated nucleic acid sequencesencoding a UDP-glucose 6-dehydrogenase selected from the groupconsisting of: (a) a nucleic acid sequence encoding a polypeptide havingan amino acid sequence which has at least about 75%, about 80%, about85%, about 90%, or about 95% identity to SEQ ID NO: 41; (b) a nucleicacid sequence having at least about 75%, about 80%, about 85%, about90%, or about 95% homology to SEQ ID NO: 40; (c) a nucleic acid sequencewhich hybridizes under medium or high stringency conditions with (i) thenucleic acid sequence of SEQ ID NO: 40, (ii) the cDNA sequence containedin SEQ ID NO: 40, or (iii) a complementary strand of (i) or (ii); and(d) a subsequence of (a), (b), or (c), wherein the subsequence encodes apolypeptide fragment which has UDP-glucose 6-dehydrogenase activity.

The present invention also relates to isolated nucleic acid sequencesencoding a UDP-glucose pyrophosphorylase selected from the groupconsisting of: (a) a nucleic acid sequence encoding a polypeptide havingan amino acid sequence which has at least about 90%, about 95%, or about97% identity to SEQ ID NO: 43; (b) a nucleic acid sequence having atleast about 90%, about 95%, or about 97% homology to SEQ ID NO: 42; (c)a nucleic acid sequence which hybridizes under low, medium, or highstringency conditions with (i) the nucleic acid sequence of SEQ ID NO:42, (ii) the cDNA sequence contained in SEQ ID NO: 42, or (iii) acomplementary strand of (i) or (ii); and (d) a subsequence of (a), (b),or (c), wherein the subsequence encodes a polypeptide fragment which hasUDP-N-acetylglucosamine pyrophosphorylase activity.

The present invention also relates to isolated nucleic acid sequencesencoding a UDP-N-acetylglucosamine pyrophosphorylase selected from thegroup consisting of: (a) a nucleic acid sequence encoding a polypeptidehaving an amino acid sequence which has at least about 75%, about 80%,about 85%, about 90%, or about 95% identity to SEQ ID NO: 45; (b) anucleic acid sequence having at least about 75%, about 80%, about 85%,about 90%, or about 95% homology to SEQ ID NO: 44; (c) a nucleic acidsequence which hybridizes under low, medium, or high stringencyconditions with (i) the nucleic acid sequence of SEQ ID NO: 44, (ii) thecDNA sequence contained in SEQ ID NO: 44, or (iii) a complementarystrand of (i) or (ii); and (d) a subsequence of (a), (b), or (c),wherein the subsequence encodes a polypeptide fragment which hasUDP-N-acetylglucosamine pyrophosphorylase activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structure of hyaluronan.

FIG. 2 shows the biosynthetic pathway for hyaluronan synthesis.

FIG. 3 shows a restriction map of pCR2.1-sehasA.

FIG. 4 shows a restriction map of pCR2.1-tuaD.

FIG. 5 shows a restriction map of pCR2.1-gtaB.

FIG. 6 shows a restriction map of pCR2.1-gcaD.

FIG. 7 shows a restriction map of pHA1.

FIG. 8 shows a restriction map of pHA2.

FIG. 9 shows a restriction map of pHA3.

FIG. 10 shows a restriction map of pHA4.

FIG. 11 shows a restriction map of pHA5.

FIG. 12 shows a restriction map of pHA6.

FIG. 13 shows a restriction map of pHA7.

FIG. 14 shows a restriction map of pMRT106.

FIG. 15 shows a restriction map of pHA8.

FIG. 16 shows a restriction map of pHA9.

FIG. 17 shows a restriction map of pHA10.

FIG. 18 shows a restriction map of pRB157.

FIG. 19 shows a restriction map of pMRT084.

FIG. 20 shows a restriction map of pMRT086.

FIG. 21 shows a restriction map of pCJ791.

FIG. 22 shows a restriction map of pMRT032.

FIG. 23 shows a restriction map of pNNB194neo.

FIG. 24 shows a restriction map of pNNB194neo-oriT.

FIG. 25 shows a restriction map of pShV3.

FIG. 26 shows a restriction map of pShV2.1-amyEΔB.

FIG. 27 shows a restriction map of pShV3A.

FIG. 28 shows a restriction map of pMRT036.

FIG. 29 shows a restriction map of pMRT037.

FIG. 30 shows a restriction map of pMRT041.

FIG. 31 shows a restriction map of pMRT064.1.

FIG. 32 shows a restriction map of pMRT068.

FIG. 33 shows a restriction map of pMRT069.

FIG. 34 shows a restriction map of pMRT071.

FIG. 35 shows a restriction map of pMRT074.

FIG. 36 shows a restriction map of pMRT120.

FIG. 37 shows a restriction map of pMRT122.

FIG. 38 shows a restriction map of pCR2.1-pel5′.

FIG. 39 shows a restriction map of pCR2.1-pel3′.

FIG. 40 shows a restriction map of pRB161.

FIG. 41 shows a restriction map of pRB162.

FIG. 42 shows a restriction map of pRB156.

FIG. 43 shows a restriction map of pRB164.

FIG. 44 shows a summary of fermentations of various hyaluronic acidproducing Bacillus subtilis strains run under fed batch at approximately2 g sucrose/L₀-hr, 37° C.

FIG. 45 shows a summary of peak hyaluronic acid weight average molecularweights (MDa) obtained from fermentations of various hyaluronic acidproducing Bacillus subtilis strains run under fed batch at approximately2 g sucrose/L₀-hr, 37° C.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for producing a hyaluronan,comprising: (a) cultivating a Bacillus host cell under conditionssuitable for production of the hyaluronan, wherein the Bacillus hostcell comprises a nucleic acid construct comprising a hyaluronan synthaseencoding sequence operably linked to a promoter sequence foreign to thehyaluronan synthase encoding sequence; and (b) recovering the hyaluronanfrom the cultivation medium.

The methods of the present invention represent an improvement over theproduction of hyaluronan from pathogenic, encapsulating bacteria. Inencapsulating bacteria, a large quantity of the hyaluronan is producedin the capsule. In processing and purifying hyaluronan from suchsources, it is first necessary to remove the hyaluronan from thecapsule, such as by the use of a surfactant, or detergent, such as SDS.This creates a complicating step in commercial production of hyaluronan,as the surfactant must be added in order to liberate a large portion ofthe hyaluronan, and subsequently the surfactant must be removed prior tofinal purification.

The present invention allows the production of a large quantity of ahyaluronan, which is produced in a non-encapsulating host cell, as freehyaluronan. When viewed under the microscope, there is no visiblecapsule associated with the recombinant strains of Bacillus, whereas thepathogenic strains traditionally used in hyaluronan production comprisea capsule of hyaluronan that is at least twice the diameter of the cellitself.

Since the hyaluronan of the recombinant Bacillus cell is expresseddirectly to the culture medium, a simple process may be used to isolatethe hyaluronan from the culture medium. First, the Bacillus cells andcellular debris are physically removed from the culture medium. Theculture medium may be diluted first, if desired, to reduce the viscosityof the medium. Many methods are known to those skilled in the art forremoving cells from culture medium, such as centrifugation ormicrofiltration. If desired, the remaining supernatant may then befiltered, such as by ultrafiltration, to concentrate and remove smallmolecule contaminants from the hyaluronan. Following removal of thecells and cellular debris, a simple precipitation of the hyaluronan fromthe medium is performed by known mechanisms. Salt, alcohol, orcombinations of salt and alcohol may be used to precipitate thehyaluronan from the filtrate. Once reduced to a precipitate, thehyaluronan can be easily isolated from the solution by physical means.Alternatively, the hyaluronan may be dried or concentrated from thefiltrate solution by using evaporative techniques known to the art, suchas spray drying.

The methods of the present invention thus represent an improvement overexisting techniques for commercially producing hyaluronan byfermentation, in not requiring the use of a surfactant in thepurification of hyaluronan from cells in culture.

Hyaluronic Acid

“Hyaluronic acid” is defined herein as an unsulphated glycosaminoglycancomposed of repeating disaccharide units of N-acetylglucosamine (GlcNAc)and glucuronic acid (GlcUA) linked together by alternating beta-1,4 andbeta-1,3 glycosidic bonds (FIG. 1). Hyaluronic acid is also known ashyaluronan, hyaluronate, or HA. The terms hyaluronan and hyaluronic acidare used interchangeably herein.

In a preferred embodiment, the hyaluronic acid obtained by the methodsof the present invention has a molecular weight of about 10,000 to about10,000,000 Da. In a more preferred embodiment, the hyaluronic acidobtained by the methods of the present invention has a molecular weightof about 25,000 to about 5,000,000 Da. In a most preferred embodiment,the hyaluronic acid obtained by the methods of the present invention hasa molecular weight of about 50,000 to about 3,000,000 Da.

The level of hyaluronic acid produced by a Bacillus host cell of thepresent invention may be determined according to the modified carbazolemethod (Bitter and Muir, 1962, Anal Biochem. 4: 330-334). Moreover, theaverage molecular weight of the hyaluronic acid may be determined usingstandard methods in the art, such as those described by Ueno et al.,1988, Chem. Pharm. Bull. 36, 4971-4975; Wyatt, 1993, Anal. Chim. Acta272: 1-40; and Wyatt Technologies, 1999, “Light Scattering UniversityDAWN Course Manual” and “DAWN EOS Manual” Wyatt Technology Corporation,Santa Barbara, Calif.

The hyaluronic acid obtained by the methods of the present invention maybe subjected to various techniques known in the art to modify thehyaluronic acid, such as crosslinking as described, for example, in U.S.Pat. Nos. 5,616,568, 5,652,347, and 5,874,417. Moreover, the molecularweight of the hyaluronic acid may be altered using techniques known inthe art.

Host Cells

In the methods of the present invention, the Bacillus host cell may beany Bacillus cell suitable for recombinant production of hyaluronicacid. The Bacillus host cell may be a wild-type Bacillus cell or amutant thereof. Bacillus cells useful in the practice of the presentinvention include, but are not limited to, Bacillus agaraderhens,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells. Mutant Bacillussubtilis cells particularly adapted for recombinant expression aredescribed in WO 98/22598. Non-encapsulating Bacillus cells areparticularly useful in the present invention.

In a preferred embodiment, the Bacillus host cell is a Bacillusamyloliquefaciens, Bacillus clausii, Bacillus lentus, Bacilluslicheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. Ina more preferred embodiment, the Bacillus cell is a Bacillusamyloliquefaciens cell. In another more preferred embodiment, theBacillus cell is a Bacillus clausii cell. In another more preferredembodiment, the Bacillus cell is a Bacillus lentus cell. In another morepreferred embodiment, the Bacillus cell is a Bacillus licheniformiscell. In another more preferred embodiment, the Bacillus cell is aBacillus subtilis cell. In a most preferred embodiment, the Bacillushost cell is Bacillus subtilis A164Δ5 (see U.S. Pat. No. 5,891,701) orBacillus subtilis 168Δ4.

Transformation of the Bacillus host cell with a nucleic acid constructof the present invention may, for instance, be effected by protoplasttransformation (see, e.g., Chang and Cohen, 1979, Molecular GeneralGenetics 168: 111-115), by using competent cells (see, e.g., Young andSpizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), byelectroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987,Journal of Bacteriology 169: 5271-5278).

Nucleic Acid Constructs

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct may besynonymous with the term expression cassette when the nucleic acidconstruct contains all the control sequences required for expression ofa coding sequence. The term “coding sequence” is defined herein as asequence which is transcribed into mRNA and translated into an enzyme ofinterest when placed under the control of the below mentioned controlsequences. The boundaries of the coding sequence are generallydetermined by a ribosome binding site located just upstream of the openreading frame at the 5′ end of the mRNA and a transcription terminatorsequence located just downstream of the open reading frame at the 3′ endof the mRNA. A coding sequence can include, but is not limited to, DNA,cDNA, and recombinant nucleic acid sequences.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are well known in the art and include, for example,isolation from genomic DNA, preparation from cDNA, or a combinationthereof. The cloning of the nucleic acid sequences from such genomic DNAcan be effected, e.g., by using antibody screening of expressionlibraries to detect cloned DNA fragments with shared structural featuresor the well known polymerase chain reaction (PCR). See, for example,Innis et al., 1990, PCR Protocols: A Guide to Methods and Application,Academic Press, New York. Other nucleic acid amplification proceduressuch as ligase chain reaction, ligated activated transcription, andnucleic acid sequence-based amplification may be used. The cloningprocedures may involve excision and isolation of a desired nucleic acidfragment comprising the nucleic acid sequence encoding the polypeptide,insertion of the fragment into a vector molecule, and incorporation ofthe recombinant vector into a Bacillus cell where clones of the nucleicacid sequence will be replicated. The nucleic acid sequence may be ofgenomic, cDNA, RNA, semi-synthetic, synthetic origin, or anycombinations thereof.

An isolated nucleic acid sequence encoding an enzyme may be manipulatedin a variety of ways to provide for expression of the enzyme.Manipulation of the nucleic acid sequence prior to its insertion into aconstruct or vector may be desirable or necessary depending on theexpression vector or Bacillus host cell. The techniques for modifyingnucleic acid sequences utilizing cloning methods are well known in theart. It will be understood that the nucleic acid sequence may also bemanipulated in vivo in the host cell using methods well known in theart.

A number of enzymes are involved in the biosynthesis of hyaluronic acid.These enzymes include hyaluronan synthase, UDP-glucose 6-dehydrogenase,UDP-glucose pyrophosphorylase, UDP-N-acetylglucosaminepyrophosphorylase, glucose-6-phosphate isomerase, hexokinase,phosphoglucomutase, amidotransferase, mutase, and acetyl transferase.Hyaluronan synthase is the key enzyme in the production of hyaluronicacid.

“Hyaluronan synthase” is defined herein as a synthase that catalyzes theelongation of a hyaluronan chain by the addition of GlcUA and GlcNAcsugar precursors. The amino acid sequences of streptococcal hyaluronansynthases, vertebrate hyaluronan synthases, and the viral hyaluronansynthase are distinct from the Pasteurella hyaluronan synthase, and havebeen proposed for classification as Group I and Group II hyaluronansynthases, the Group I hyaluronan synthases including Streptococcalhyaluronan synthases (DeAngelis, 1999). For production of hyaluronan inBacillus host cells, hyaluronan synthases of a eukaryotic origin, suchas mammalian hyaluronan synthases, are less preferred.

The hyaluronan synthase encoding sequence may be any nucleic acidsequence capable of being expressed in a Bacillus host cell. The nucleicacid sequence may be of any origin. Preferred hyaluronan synthase genesinclude any of either Group I or Group II, such as the Group Ihyaluronan synthase genes from Streptococcus equisimilis, Streptococcuspyogenes, Streptococcus uberis, and Streptococcus equi subsp.zooepidemicus, or the Group II hyaluronan synthase genes of Pasturellamultocida.

In a preferred embodiment, the hyaluronan synthase encoding sequence isselected from the group consisting of (a) a nucleic acid sequenceencoding a polypeptide with an amino acid sequence having at least about70%, about 75%, about 80%, about 85%, about 90%, or about 95% identityto SEQ ID NO: 2, SEQ ID NO: 93, or SEQ ID NO: 103; (b) a nucleic acidsequence which hybridizes under low, medium, or high stringencyconditions with SEQ ID NO: 1, SEQ ID NO: 92, or SEQ ID NO: 102; and (c)a complementary strand of (a) or (b).

In a more preferred embodiment, the hyaluronan synthase encodingsequence encodes a polypeptide having the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 93, or SEQ ID NO: 103; or a fragment thereof havinghyaluronan synthase activity.

In another preferred embodiment, the hyaluronan synthase encodingsequence is selected from the group consisting of (a) a nucleic acidsequence encoding a polypeptide with an amino acid sequence having atleast about 70%, about 75%, about 80%, about 85%, about 90%, or about95% identity to SEQ ID NO: 95; (b) a nucleic acid sequence whichhybridizes under low, medium, or high stringency conditions with SEQ IDNO: 94; and (c) a complementary strand of (a) or (b).

In another more preferred embodiment, the hyaluronan synthase encodingsequence encodes a polypeptide having the amino acid sequence of SEQ IDNO: 95, or a fragment thereof having hyaluronan synthase activity.

The methods of the present invention also include constructs wherebyprecursor sugars of hyaluronan are supplied to the host cell, either tothe culture medium, or by being encoded by endogenous genes, bynon-endogenous genes, or by a combination of endogenous andnon-endogenous genes in the Bacillus host cell. The precursor sugar maybe D-glucuronic acid or N-acetyl-glucosamine.

In the methods of the present invention, the nucleic acid construct mayfurther comprise one or more genes encoding enzymes in the biosynthesisof a precursor sugar of a hyaluronan. Alternatively, the Bacillus hostcell may further comprise one or more second nucleic acid constructscomprising one or more genes encoding enzymes in the biosynthesis of theprecursor sugar. Hyaluronan production is improved by the use ofconstructs with a nucleic acid sequence or sequences encoding a gene orgenes directing a step in the synthesis pathway of the precursor sugarof hyaluronan. By, “directing a step in the synthesis pathway of aprecursor sugar of hyaluronan” is meant that the expressed protein ofthe gene is active in the formation of N-acetyl-glucosamine orD-glucuronic acid, or a sugar that is a precursor of either ofN-acetyl-glucosamine and D-glucuronic acid (FIG. 2).

In a preferred method for supplying precursor sugars, constructs areprovided for improving hyaluronan production in a host cell having ahyaluronan synthase, by culturing a host cell having a recombinantconstruct with a heterologous promoter region operably linked to anucleic acid sequence encoding a gene directing a step in the synthesispathway of a precursor sugar of hyaluronan. In a preferred method thehost cell also comprises a recombinant construct having a promoterregion operably linked to a hyaluronan synthase, which may use the sameor a different promoter region than the nucleic acid sequence to asynthase involved in the biosynthesis of N-acetyl-glucosamine. In afurther preferred embodiment, the host cell may have a recombinantconstruct with a promoter region operably linked to different nucleicacid sequences encoding a second gene involved in the synthesis of aprecursor sugar of hyaluronan.

Thus, the present invention also relates to constructs for improvinghyaluronan production by the use of constructs with a nucleic acidsequence encoding a gene directing a step in the synthesis pathway of aprecursor sugar of hyaluronan. The nucleic acid sequence to theprecursor sugar may be expressed from the same or a different promoteras the nucleic acid sequence encoding the hyaluronan synthase.

The genes involved in the biosynthesis of precursor sugars for theproduction of hyaluronic acid include a UDP-glucose 6-dehydrogenasegene, UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosaminepyrophosphorylase gene, glucose-6-phosphate isomerase gene, hexokinasegene, phosphoglucomutase gene, amidotransferase gene, mutase gene, andacetyl transferase gene.

In a cell containing a hyaluronan synthase, any one or combination oftwo or more of hasB, hasC and hasD, or the homologs thereof, such as theBacillus subtilis tuaD, gtaB, and gcaD, respectively, as well as hasE,may be expressed to increase the pools of precursor sugars available tothe hyaluronan synthase. The Bacillus genome is described in Kunst, etal., Nature 390, 249-256, “The complete genome sequence of theGram-positive bacterium Bacillus subtilis” (20 Nov. 1997). In someinstances, such as where the host cell does not have a native hyaluronansynthase activity, the construct may include the hasA gene.

The nucleic acid sequence encoding the biosynthetic enzymes may benative to the host cell, while in other cases heterologous sequence maybe utilized. If two or more genes are expressed they may be genes thatare associated with one another in a native operon, such as the genes ofthe HAS operon of Streptococcus equisimilis, which comprises hasA, hasB,hasC and hasD. In other instances, the use of some combination of theprecursor gene sequences may be desired, without each element of theoperon included. The use of some genes native to the host cell, andothers which are exogenous may also be preferred in other cases. Thechoice will depend on the available pools of sugars in a given hostcell, the ability of the cell to accommodate overproduction withoutinterfering with other functions of the host cell, and whether the cellregulates expression from its native genes differently than exogenousgenes.

As one example, depending on the metabolic requirements and growthconditions of the cell, and the available precursor sugar pools, it maybe desirable to increase the production of N-acetyl-glucosamine byexpression of a nucleic acid sequence encoding UDP-N-acetylglucosaminepyrophosphorylase, such as the hasD gene, the Bacillus gcaD gene, andhomologs thereof. Alternatively, the precursor sugar may be D-glucuronicacid. In one such embodiment, the nucleic acid sequence encodesUDP-glucose 6-dehydrogenase. Such nucleic acid sequences include theBacillus tuaD gene, the hasB gene of Streptococcus, and homologsthereof. The nucleic acid sequence may also encode UDP-glucosepyrophosphorylase, such as in the Bacillus gtaB gene, the hasC gene ofStreptococcus, and homologs thereof.

In the methods of the present invention, the UDP-glucose 6-dehydrogenasegene may be a hasB gene or tuaD gene; or homologs thereof.

In a preferred embodiment, the hasB gene is selected from the groupconsisting of (a) a nucleic acid sequence encoding a polypeptide with anamino acid sequence having at least about 70%, about 75%, about 80%,about 85%, about 90%, or about 95% identity to SEQ ID NO: 41, SEQ ID NO:97, or SEQ ID NO: 105; (b) a nucleic acid sequence which hybridizesunder low, medium, or high stringency conditions with SEQ ID NO: 40, SEQID NO: 96, or SEQ ID NO: 104; and (c) a complementary strand of (a) or(b).

In a more preferred embodiment, the hasB gene encodes a polypeptidehaving the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 97, or SEQID NO: 105; or a fragment thereof having UDP-glucose 6-dehydrogenaseactivity.

In another preferred embodiment, the tuaD gene is selected from thegroup consisting of (a) a nucleic acid sequence encoding a polypeptidewith an amino acid sequence having at least about 70%, about 75%, about80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 12; (b) anucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 11; and (c) a complementary strandof (a) or (b).

In another more preferred embodiment, the tuaD gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 12, or afragment thereof having UDP-glucose 6-dehydrogenase activity.

In the methods of the present invention, the UDP-glucosepyrophosphorylase gene may be a hasC gene or gtaB gene; or homologsthereof.

In a preferred embodiment, the hasC gene is selected from the groupconsisting of (a) a nucleic acid sequence encoding a polypeptide with anamino acid sequence having at least about 70%, about 75%, about 80%,about 85%, about 90%, or about 95% identity to SEQ ID NO: 43, SEQ ID NO:99, or SEQ ID NO: 107; (b) a nucleic acid sequence which hybridizesunder low, medium, or high stringency conditions with SEQ ID NO: 42 orSEQ ID NO: 98, or SEQ ID NO: 106; and (c) a complementary strand of (a)or (b).

In another more preferred embodiment, the hasC gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 43 or SEQ IDNO: 99, or SEQ ID NO: 107; or a fragment thereof having UDP-glucosepyrophosphorylase activity.

In another preferred embodiment, the gtaB gene is selected from thegroup consisting of (a) a nucleic acid sequence encoding a polypeptidewith an amino acid sequence having at least about 70%, about 75%, about80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 22; (b) anucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 21; and (c) a complementary strandof (a) or (b).

In another more preferred embodiment, the gtaB gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 22, or afragment thereof having UDP-glucose pyrophosphorylase activity.

In the methods of the present invention, the UDP-N-acetylglucosaminepyrophosphorylase gene may be a hasD or gcaD gene; or homologs thereof.

In a preferred embodiment, the hasD gene is selected from the groupconsisting of (a) a nucleic acid sequence encoding a polypeptide with anamino acid sequence having at least about 75%, about 80%, about 85%,about 90%, or about 95% identity to SEQ ID NO: 45; (b) a nucleic acidsequence which hybridizes under low, medium, or high stringencyconditions with SEQ ID NO: 44; and (c) a complementary strand of (a) or(b).

In another more preferred embodiment, the hasD gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 45, or afragment thereof having UDP-N-acetylglucosamine pyrophosphorylaseactivity.

In another preferred embodiment, the gcaD gene is selected from thegroup consisting of (a) a nucleic acid sequence encoding a polypeptidewith an amino acid sequence having at least about 70%, about 75%, about80%, about 85%, about 90%, or about 95% identity to SEQ ID NO: 30; (b) anucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 29; and (c) a complementary strandof (a) or (b).

In another more preferred embodiment, the gcaD gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 30, or afragment thereof having UDP-N-acetylglucosamine pyrophosphorylaseactivity.

In the methods of the present invention, the glucose-6-phosphateisomerase gene may be a hasE or homolog thereof.

In a preferred embodiment, the hasE gene is selected from the groupconsisting of (a) a nucleic acid sequence encoding a polypeptide with anamino acid sequence having at least about 70%, about 75%, about 80%,about 85%, about 90%, or about 95% identity to SEQ ID NO: 101; (b) anucleic acid sequence which hybridizes under low, medium, or highstringency conditions with SEQ ID NO: 100; and (c) a complementarystrand of (a) or (b).

In another more preferred embodiment, the hasE gene encodes apolypeptide having the amino acid sequence of SEQ ID NO: 101, or afragment thereof having glucose-6-phosphate isomerase activity.

In the methods of the present invention, the hyaluronan synthase geneand the one or more genes encoding a precursor sugar are under thecontrol of the same promoter. Alternatively, the one or more genesencoding a precursor sugar are under the control of the same promoterbut a different promoter driving the hyaluronan synthase gene. A furtheralternative is that the hyaluronan synthase gene and each of the genesencoding a precursor sugar are under the control of different promoters.In a preferred embodiment, the hyaluronan synthase gene and the one ormore genes encoding a precursor sugar are under the control of the samepromoter.

The present invention also relates to a nucleic acid constructcomprising an isolated nucleic acid sequence encoding a hyaluronansynthase operon comprising a hyaluronan synthase gene and a UDP-glucose6-dehydrogenase gene, and optionally one or more genes selected from thegroup consisting of a UDP-glucose pyrophosphorylase gene,UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphateisomerase gene. A nucleic acid sequence encoding most of the hyaluronansynthase operon of Streptococcus equisimilis is found in SEQ ID NO: 108.This sequence contains the hasB (SEQ ID NO: 40) and hasC (SEQ ID nO: 42)homologs of the Bacillus subtilis tuaD gene (SEQ ID NO: 11) and gtaBgene (SEQ ID NO: 21), respectively, as is the case for Streptococcuspyogenes, as well as a homolog of the gcaD gene (SEQ ID NO: 29), whichhas been designated hasD (SEQ ID NO: 44). The Bacillus subtilis gcaDencodes UDP-N-acetylglucosamine pyrophosphorylase, which is involved inthe synthesis of N-acetyl-glucosamine, one of the two sugars ofhyaluronan. The Streptococcus equisimilis homolog of gcaD, hasD, isarranged by Streptococcus equisimilis on the hyaluronan synthase operon.The nucleic aci sequence also contains a portion of the hasA gene (thelast 1156 bp of SEQ ID NO: 1).

In some cases the host cell will have a recombinant construct with aheterologous promoter region operably linked to a nucleic acid sequenceencoding a gene directing a step in the synthesis pathway of a precursorsugar of hyaluronan, which may be in concert with the expression ofhyaluronan synthase from a recombinant construct. The hyaluronansynthase may be expressed from the same or a different promoter regionthan the nucleic acid sequence encoding an enzyme involved in thebiosynthesis of the precursor. In another preferred embodiment, the hostcell may have a recombinant construct with a promoter region operablylinked to a different nucleic acid sequence encoding a second geneinvolved in the synthesis of a precursor sugar of hyaluronan.

The nucleic acid sequence encoding the enzymes involved in thebiosynthesis of the precursor sugar(s) may be expressed from the same ora different promoter as the nucleic acid sequence encoding thehyaluronan synthase. In the former sense, “artificial operons” areconstructed, which may mimic the operon of Streptococcus equisimilis inhaving each hasA, hasB, hasC and hasD, or homologs thereof, or,alternatively, may utilize less than the full complement present in theStreptococcus equisimilis operon. The artificial operons” may alsocomprise a glucose-6-phosphate isomerase gene (hasE) as well as one ormore genes selected from the group consisting of a hexokinase gene,phosphoglucomutase gene, amidotransferase gene, mutase gene, and acetyltransferase gene. In the artificial operon, at least one of the elementsis heterologous to one other of the elements, such as the promoterregion being heterologous to the encoding sequences.

In a preferred embodiment, the nucleic acid construct comprises hasA,tuaD, and gtaB. In another preferred embodiment, the nucleic acidconstruct comprises hasA, tuaD, gtaB, and gcaD. In another preferredembodiment, the nucleic acid construct comprises hasA and tuaD. Inanother preferred embodiment, the nucleic acid construct comprises hasA.In another preferred embodiment, the nucleic acid construct compriseshasA, tuaD, gtaB, gcaD, and hasE. In another preferred embodiment, thenucleic acid construct comprises hasA, hasB, hasC, and hasD. In anotherpreferred embodiment, the nucleic acid construct comprises hasA, hasB,hasC, hasD, and hasE. Based on the above preferred embodiments, thegenes noted can be replaced with homologs thereof.

In the methods of the present invention, the nucleic acid constructscomprise a hyaluronan synthase encoding sequence operably linked to apromoter sequence foreign to the hyaluronan synthase encoding sequence.The promoter sequence may be, for example, a single promoter or a tandempromoter.

“Promoter” is defined herein as a nucleic acid sequence involved in thebinding of RNA polymerase to initiate transcription of a gene. “Tandempromoter” is defined herein as two or more promoter sequences each ofwhich is operably linked to a coding sequence and mediates thetranscription of the coding sequence into mRNA. “Operably linked” isdefined herein as a configuration in which a control sequence, e.g., apromoter sequence, is appropriately placed at a position relative to acoding sequence such that the control sequence directs the production ofa polypeptide encoded by the coding sequence. As noted earlier, a“coding sequence” is defined herein as a nucleic acid sequence which istranscribed into mRNA and translated into a polypeptide when placedunder the control of the appropriate control sequences. The boundariesof the coding sequence are generally determined by a ribosome bindingsite located just upstream of the open reading frame at the 5′ end ofthe mRNA and a transcription terminator sequence located just downstreamof the open reading frame at the 3′ end of the mRNA. A coding sequencecan include, but is not limited to, genomic DNA, cDNA, semisynthetic,synthetic, and recombinant nucleic acid sequences.

In a preferred embodiment, the promoter sequences may be obtained from abacterial source. In a more preferred embodiment, the promoter sequencesmay be obtained from a gram positive bacterium such as a Bacillusstrain, e.g., Bacillus agaradherens, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis; or a Streptomyces strain, e.g., Streptomyces lividans orStreptomyces murinus; or from a gram negative bacterium, e.g., E. colior Pseudomonas sp.

Examples of suitable promoters for directing the transcription of anucleic acid sequence in the methods of the present invention are thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus lentus or Bacillus clausii alkalineprotease gene (aprH), Bacillus licheniformis alkaline protease gene(subtilisin Carlsberg gene), Bacillus subtilis levansucrase gene (sacB),Bacillus subtilis alpha-amylase gene (amyE), Bacillus licheniformisalpha-amylase gene (amyL), Bacillus stearothermophilus maltogenicamylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene(amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillussubtilis xylA and xylB genes, Bacillus thuringiensis subsp. tenebrionisCryIIIA gene (cryIIIA) or portions thereof, prokaryotic beta-lactamasegene (VIIIa-Kamaroff et al., 1978, Proceedings of the National Academyof Sciences USA 75:3727-3731). Other examples are the promoter of thespo1 bacterial phage promoter and the tac promoter (DeBoer et al., 1983,Proceedings of the National Academy of Sciences USA 80:21-25). Furtherpromoters are described in “Useful proteins from recombinant bacteria”in Scientific American, 1980, 242:74-94; and in Sambrook, Fritsch, andManiatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.

The promoter may also be a “consensus” promoter having the sequenceTTGACA for the “−35” region and TATAAT for the “−10” region. Theconsensus promoter may be obtained from any promoter which can functionin a Bacillus host cell. The construction of a “consensus” promoter maybe accomplished by site-directed mutagenesis to create a promoter whichconforms more perfectly to the established consensus sequences for the“−10” and “−35” regions of the vegetative “sigma A-type” promoters forBacillus subtilis (Voskuil et al., 1995, Molecular Microbiology 17:271-279).

In a preferred embodiment, the “consensus” promoter is obtained from apromoter obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus clausii or Bacillus lentus alkalineprotease gene (aprH), Bacillus licheniformis alkaline protease gene(subtilisin Carlsberg gene), Bacillus subtilis levansucrase gene (sacB),Bacillus subtilis alpha-amylase gene (amyE), Bacillus licheniformisalpha-amylase gene (amyL), Bacillus stearothermophilus maltogenicamylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene(amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillussubtilis xylA and xylB genes, Bacillus thuringiensis subsp. tenebrionisCryIIIA gene (cryIIIA) or portions thereof, or prokaryoticbeta-lactamase gene spol bacterial phage promoter. In a more preferredembodiment, the “consensus” promoter is obtained from Bacillusamyloliquefaciens alpha-amylase gene (amyQ).

Widner, et al., U.S. Pat. Nos. 6,255,076 and 5,955,310, describe tandempromoters and constructs and methods for use in expression in Bacilluscells, including the short consensus amyQ promoter (also called scBAN).The use of the cryIIIA stabilizer sequence, and constructs using thesequence, for improved production in Bacillus are also describedtherein.

Each promoter sequence of the tandem promoter may be any nucleic acidsequence which shows transcriptional activity in the Bacillus cell ofchoice including a mutant, truncated, and hybrid promoter, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the Bacillus cell. Each promotersequence may be native or foreign to the nucleic acid sequence encodingthe polypeptide and native or foreign to the Bacillus cell. The promotersequences may be the same promoter sequence or different promotersequences.

The two or more promoter sequences of the tandem promoter maysimultaneously promote the transcription of the nucleic acid sequence.Alternatively, one or more of the promoter sequences of the tandempromoter may promote the transcription of the nucleic acid sequence atdifferent stages of growth of the Bacillus cell.

In a preferred embodiment, the tandem promoter contains at least theamyQ promoter of the Bacillus amyloliquefaciens alpha-amylase gene. Inanother preferred embodiment, the tandem promoter contains at least a“consensus” promoter having the sequence TTGACA for the “−35” region andTATAAT for the “−10” region. In another preferred embodiment, the tandempromoter contains at least the amyL promoter of the Bacilluslicheniformis alpha-amylase gene. In another preferred embodiment, thetandem promoter contains at least the cryIIIA promoter or portionsthereof (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107).

In a more preferred embodiment, the tandem promoter contains at leastthe amyL promoter and the cryIIIA promoter. In another more preferredembodiment, the tandem promoter contains at least the amyQ promoter andthe cryIIIA promoter. In another more preferred embodiment, the tandempromoter contains at least a “consensus” promoter having the sequenceTTGACA for the “−35” region and TATAAT for the “−10” region and thecryIIIA promoter. In another more preferred embodiment, the tandempromoter contains at least two copies of the amyL promoter. In anothermore preferred embodiment, the tandem promoter contains at least twocopies of the amyQ promoter. In another more preferred embodiment, thetandem promoter contains at least two copies of a “consensus” promoterhaving the sequence TTGACA for the “−35” region and TATAAT for the “−10”region. In another more preferred embodiment, the tandem promotercontains at least two copies of the cryIIIA promoter.

“An mRNA processing/stabilizing sequence” is defined herein as asequence located downstream of one or more promoter sequences andupstream of a coding sequence to which each of the one or more promotersequences are operably linked such that all mRNAs synthesized from eachpromoter sequence may be processed to generate mRNA transcripts with astabilizer sequence at the 5′ end of the transcripts. The presence ofsuch a stabilizer sequence at the 5′ end of the mRNA transcriptsincreases their half-life (Agaisse and Lereclus, 1994, supra, Hue etal., 1995, Journal of Bacteriology 177: 3465-3471). The mRNAprocessing/stabilizing sequence is complementary to the 3′ extremity ofa bacterial 16S ribosomal RNA. In a preferred embodiment, the mRNAprocessing/stabilizing sequence generates essentially single-sizetranscripts with a stabilizing sequence at the 5′ end of thetranscripts. The mRNA processing/stabilizing sequence is preferably one,which is complementary to the 3′ extremity of a bacterial 16S ribosomalRNA. See, U.S. Pat. Nos. 6,255,076 and 5,955,310.

In a more preferred embodiment, the mRNA processing/stabilizing sequenceis the Bacillus thuringiensis cryIIIA mRNA processing/stabilizingsequence disclosed in WO 94/25612 and Agaisse and Lereclus, 1994, supra,or portions thereof which retain the mRNA processing/stabilizingfunction. In another more preferred embodiment, the mRNAprocessing/stabilizing sequence is the Bacillus subtilis SP82 mRNAprocessing/stabilizing sequence disclosed in Hue et al., 1995, supra, orportions thereof which retain the mRNA processing/stabilizing function.

When the cryIIIA promoter and its mRNA processing/stabilizing sequenceare employed in the methods of the present invention, a DNA fragmentcontaining the sequence disclosed in WO 94/25612 and Agaisse andLereclus, 1994, supra, or portions thereof which retain the promoter andmRNA processing/stabilizing functions, may be used. Furthermore, DNAfragments containing only the cryIIIA promoter or only the cryIIIA mRNAprocessing/stabilizing sequence may be prepared using methods well knownin the art to construct various tandem promoter and mRNAprocessing/stabilizing sequence combinations. In this embodiment, thecryIIIA promoter and its mRNA processing/stabilizing sequence arepreferably placed downstream of the other promoter sequence(s)constituting the tandem promoter and upstream of the coding sequence ofthe gene of interest.

The isolated nucleic acid sequence encoding the desired enzyme(s)involved in hyaluronic acid production may then be further manipulatedto improve expression of the nucleic acid sequence. Expression will beunderstood to include any step involved in the production of thepolypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion. The techniques for modifying nucleic acidsequences utilizing cloning methods are well known in the art.

A nucleic acid construct comprising a nucleic acid sequence encoding anenzyme may be operably linked to one or more control sequences capableof directing the expression of the coding sequence in a Bacillus cellunder conditions compatible with the control sequences.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for expression of the codingsequence of a nucleic acid sequence. Each control sequence may be nativeor foreign to the nucleic acid sequence encoding the enzyme. In additionto promoter sequences described above, such control sequences include,but are not limited to, a leader, a signal sequence, and a transcriptionterminator. At a minimum, the control sequences include a promoter, andtranscriptional and translational stop signals. The control sequencesmay be provided with linkers for the purpose of introducing specificrestriction sites facilitating ligation of the control sequences withthe coding region of the nucleic acid sequence encoding an enzyme.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a Bacillus cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the enzyme or the lastenzyme of an operon. Any terminator which is functional in the Bacilluscell of choice may be used in the present invention.

The control sequence may also be a suitable leader sequence, anontranslated region of a mRNA which is important for translation by theBacillus cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the enzyme. Any leader sequencewhich is functional in the Bacillus cell of choice may be used in thepresent invention.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of apolypeptide which can direct the expressed polypeptide into the cell'ssecretory pathway. The signal peptide coding region may be native to thepolypeptide or may be obtained from foreign sources. The 5′ end of thecoding sequence of the nucleic acid sequence may inherently contain asignal peptide coding region naturally linked in translation readingframe with the segment of the coding region which encodes the secretedpolypeptide. Alternatively, the 5′ end of the coding sequence maycontain a signal peptide coding region which is foreign to that portionof the coding sequence which encodes the secreted polypeptide. Theforeign signal peptide coding region may be required where the codingsequence does not normally contain a signal peptide coding region.Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to obtainenhanced secretion of the polypeptide relative to the natural signalpeptide coding region normally associated with the coding sequence. Thesignal peptide coding region may be obtained from an amylase or aprotease gene from a Bacillus species. However, any signal peptidecoding region capable of directing the expressed polypeptide into thesecretory pathway of a Bacillus cell of choice may be used in thepresent invention.

An effective signal peptide coding region for Bacillus cells is thesignal peptide coding region obtained from the maltogenic amylase genefrom Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylasegene, the Bacillus licheniformis subtilisin gene, the Bacilluslicheniformis beta-lactamase gene, the Bacillus stearothermophilusneutral proteases genes (nprT, nprS, nprM), and the Bacillus subtilisprsA gene. Further signal peptides are described by Simonen and Palva,1993, Microbiological Reviews 57:109-137.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE) and Bacillussubtilis neutral protease (nprT).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems.

Expression Vectors

In the methods of the present invention, a recombinant expression vectorcomprising a nucleic acid sequence, a promoter, and transcriptional andtranslational stop signals may be used for the recombinant production ofan enzyme involved in hyaluronic acid production. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide or enzyme at suchsites. Alternatively, the nucleic acid sequence may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression, and possibly secretion.

The recombinant expression vector may be any vector which can beconveniently subjected to recombinant DNA procedures and can bring aboutthe expression of the nucleic acid sequence. The choice of the vectorwill typically depend on the compatibility of the vector with theBacillus cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids. The vector may be an autonomouslyreplicating vector, i.e., a vector which exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Alternatively, the vector may beone which, when introduced into the Bacillus cell, is integrated intothe genome and replicated together with the chromosome(s) into which ithas been integrated. The vector system may be a single vector or plasmidor two or more vectors or plasmids which together contain the total DNAto be introduced into the genome of the Bacillus cell, or a transposonmay be used.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the Bacillus host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for integration of the vector into the genome by homologousor nonhomologous recombination. Alternatively, the vector may containadditional nucleic acid sequences for directing integration byhomologous recombination into the genome of the Bacillus cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the Bacillus cell genome at a precise location in the chromosome.To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the Bacillus cell. Furthermore, the integrational elements maybe non-encoding or encoding nucleic acid sequences. On the other hand,the vector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in theBacillus cell in question. Examples of bacterial origins of replicationare the origins of replication of plasmids pUB110, pE194, pTA1060, andpAMβ1 permitting replication in Bacillus. The origin of replication maybe one having a mutation to make its function temperature-sensitive inthe Bacillus cell (see, e.g., Ehrlich, 1978, Proceedings of the NationalAcademy of Sciences USA 75:1433).

The vectors preferably contain one or more selectable markers whichpermit easy selection of transformed cells. A selectable marker is agene the product of which provides for biocide resistance, resistance toheavy metals, prototrophy to auxotrophs, and the like. Examples ofbacterial selectable markers are the dal genes from Bacillus subtilis orBacillus licheniformis, or markers which confer antibiotic resistancesuch as ampicillin, kanamycin, chloramphenicol or tetracyclineresistance. Furthermore, selection may be accomplished byco-transformation, e.g., as described in WO 91/09129, where theselectable marker is on a separate vector.

More than one copy of a nucleic acid sequence may be inserted into thehost cell to increase production of the gene product. An increase in thecopy number of the nucleic acid sequence can be obtained by integratingat least one additional copy of the sequence into the host cell genomeor by including an amplifiable selectable marker gene with the nucleicacid sequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the nucleic acid sequence,can be selected for by cultivating the cells in the presence of theappropriate selectable agent. A convenient method for achievingamplification of genomic DNA sequences is described in WO 94/14968.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

Production

In the methods of the present invention, the Bacillus host cells arecultivated in a nutrient medium suitable for production of thehyaluronic acid using methods known in the art. For example, the cellmay be cultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the enzymes involved inhyaluronic acid synthesis to be expressed and the hyaluronic acid to beisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). The secreted hyaluronic acid can be recovered directly fromthe medium.

The resulting hyaluronic acid may be isolated by methods known in theart. For example, the hyaluronic acid may be isolated from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. The isolated hyaluronic acid may then be further purifiedby a variety of procedures known in the art including, but not limitedto, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

In the methods of the present invention, the Bacillus host cells producegreater than about 4 g, preferably greater than about 6 g, morepreferably greater than about 8 g, even more preferably greater thanabout 10 g, and most preferably greater than about 12 g of hyaluronicacid per liter.

Deletions/Disruptions

Gene deletion or replacement techniques may be used for the completeremoval of a selectable marker gene or other undesirable gene. In suchmethods, the deletion of the selectable marker gene may be accomplishedby homologous recombination using a plasmid that has been constructed tocontiguously contain the 5′ and 3′ regions flanking the selectablemarker gene. The contiguous 5′ and 3′ regions may be introduced into aBacillus cell on a temperature-sensitive plasmid, e.g., pE194, inassociation with a second selectable marker at a permissive temperatureto allow the plasmid to become established in the cell. The cell is thenshifted to a non-permissive temperature to select for cells that havethe plasmid integrated into the chromosome at one of the homologousflanking regions. Selection for integration of the plasmid is effectedby selection for the second selectable marker. After integration, arecombination event at the second homologous flanking region isstimulated by shifting the cells to the permissive temperature forseveral generations without selection. The cells are plated to obtainsingle colonies and the colonies are examined for loss of bothselectable markers (see, for example, Perego, 1993, In A. L. Sonneshein,J. A. Hoch, and R. Losick, editors, Bacillus subtilis and OtherGram-Positive Bacteria, Chapter 42, American Society of Microbiology,Washington, D.C., 1993).

A selectable marker gene may also be removed by homologous recombinationby introducing into the mutant cell a nucleic acid fragment comprising5′ and 3′ regions of the defective gene, but lacking the selectablemarker gene, followed by selecting on the counter-selection medium. Byhomologous recombination, the defective gene containing the selectablemarker gene is replaced with the nucleic acid fragment lacking theselectable marker gene. Other methods known in the art may also be used.

U.S. Pat. No. 5,891,701 discloses techniques for deleting several genesincluding spollAC, aprE, nprE, and amyE.

Other undesirable biological compounds may also be removed by the abovedescribed methods such as the red pigment synthesized by cypX (accessionno. BG12580) and/or yvmC (accession no. BG14121).

In a preferred embodiment, the Bacillus host cell is unmarked with anyheterologous or exogenous selectable markers. In another preferredembodiment, the Bacillus host cell does not produce any red pigmentsynthesized by cypX and yvmC.

Isolated Nucleic Acid Sequences Encoding Polypeptides Having UDP-Glucose6-Dehydrogenase Activity, UDP-Glucose Pyrophosphorylase Activity, orUDP-N-Acetylglucosamine Pyrophosphorylase Activity

The term “UDP-glucose 6-dehydrogenase activity” is defined herein as aUDP glucose:NAD⁺ 6-oxidoreductase activity which catalyzes theconversion of UDP-glucose in the presence of 2NAD⁺ and water toUDP-glucuronate and 2NADH. For purposes of the present inventionUDP-glucose 6-dehydrogenase activity is determined according to theprocedure described by Jaenicke and Rudolph, 1986, Biochemistry 25:7283-7287. One unit of UDP-glucose 6-dehydrogenase activity is definedas 1.0 μmole of UDP-glucuronate produced per minute at 25° C., pH 7.

The term “UDP-glucose pyrophosphorylase activity” is defined herein as aUTP:□-D-glucose-1-phosphate uridylyltransferase activity which catalyzesthe conversion of glucose-1-phosphate in the presence of UTP todiphosphate and UDP-glucose. For purposes of the present inventionUDP-glucose pyrophosphorylase activity is determined according to theprocedure described by Kamogawa et al., 1965, J. Biochem. (Tokyo) 57:758-765 or Hansen et al., 1966, Method Enzymol. 8: 248-253. One unit ofUDP-glucose pyrophosphorylase activity is defined as 1.0 μmole ofUDP-glucose produced per minute at 25° C., pH 7.

The term “UDP-N-acetylglucosamine pyrophosphorylase activity” is definedherein as a UTP:N-acetyl-alpha-D-glucoamine-1-phosphateuridyltransferase activity which catalyzes the conversion ofN-acetyl-alpha-D-glucosamine-1-phosphate in the presence of UTP todiphosphate and UDP-N-acetyl-alpha-D-glucoamine. For purposes of thepresent invention, UDP-N-acetylglucosamine pyrophosphorylase activity isdetermined according to the procedure described by Mangin-Lecreuix etal., 1994, J. Bacteriology 176: 5788-5795. One unit ofUDP-N-acetylglucosamine pyrophosphorylase activity is defined as 1.0mmole of UDP-N-acetyl-alpha-D-glucoamine produced per minute at 25° C.,pH 7.

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

In a first embodiment, the present invention relates to isolated nucleicacid sequences encoding polypeptides having an amino acid sequence whichhas a degree of identity to SEQ ID NO: 41 of at least about 75%,preferably at least about 80%, more preferably at least about 85%, evenmore preferably at least about 90%, most preferably at least about 95%,and even most preferably at least about 97%, which have UDP-glucose6-dehydrogenase activity (hereinafter “homologous polypeptides”). In apreferred embodiment, the homologous polypeptides have an amino acidsequence which differs by five amino acids, preferably by four aminoacids, more preferably by three amino acids, even more preferably by twoamino acids, and most preferably by one amino acid from SEQ ID NO: 41.

In another first embodiment, the present invention relates to isolatednucleic acid sequences encoding polypeptides having an amino acidsequence which has a degree of identity to SEQ ID NO: 43 of at leastabout 90%, preferably at least about 95%, and more preferably at leastabout 97%, which have UDP-glucose pyrophosphorylase activity(hereinafter “homologous polypeptides”). In a preferred embodiment, thehomologous polypeptides have an amino acid sequence which differs byfive amino acids, preferably by four amino acids, more preferably bythree amino acids, even more preferably by two amino acids, and mostpreferably by one amino acid from SEQ ID NO: 43.

In another first embodiment, the present invention relates to isolatednucleic acid sequences encoding polypeptides having an amino acidsequence which has a degree of identity to SEQ ID NO: 45 of at leastabout 75%, preferably at least about 80%, more preferably at least about85%, even more preferably at least about 90%, most preferably at leastabout 95%, and even most preferably at least about 97%, which haveUDP-N-acetylglucosamine pyrophosphorylase activity (hereinafter“homologous polypeptides”). In a preferred embodiment, the homologouspolypeptides have an amino acid sequence which differs by five aminoacids, preferably by four amino acids, more preferably by three aminoacids, even more preferably by two amino acids, and most preferably byone amino acid from SEQ ID NO: 45.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by the Clustal method (Higgins,1989, CABIOS 5: 151-153) using the Vector NTI AlignX software package(Informax Inc., Bethesda, Md.) with the following defaults: pairwisealignment, gap opening penalty of 10, gap extension penalty of 0.1, andscore matrix: blosum62mt2.

Preferably, the nucleic acid sequences of the present invention encodepolypeptides that comprise the amino acid sequence of SEQ ID NO: 41, SEQID NO: 43, or SEQ ID NO: 45; or an allelic variant thereof; or afragment thereof that has UDP-glucose 6-dehydrogenase, UDP-glucosepyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylaseactivity, respectively. In a more preferred embodiment, the nucleic acidsequence of the present invention encodes a polypeptide that comprisesthe amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO:45. In another preferred embodiment, the nucleic acid sequence of thepresent invention encodes a polypeptide that consists of the amino acidsequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45; or anallelic variant thereof; or a fragment thereof, wherein the polypeptidefragment has UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase,or UDP-N-acetylglucosamine pyrophosphorylase activity, respectively. Inanother preferred embodiment, the nucleic acid sequence of the presentinvention encodes a polypeptide that consists of the amino acid sequenceof SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45.

The present invention also encompasses nucleic acid sequences whichencode a polypeptide having the amino acid sequence of SEQ ID NO: 41,SEQ ID NO: 43, or SEQ ID NO: 45, which differ from SEQ ID NO: 40, SEQ IDNO: 42, or SEQ ID NO: 44 bp virtue of the degeneracy of the geneticcode. The present invention also relates to subsequences of SEQ ID NO:40, SEQ ID NO: 42, or SEQ ID NO: 44 which encode fragments of SEQ ID NO:41, SEQ ID NO: 43, or SEQ ID NO: 45, respectively, which haveUDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, orUDP-N-acetylglucosamine pyrophosphorylase activity, respectively.

A subsequence of SEQ ID NO: 40 is a nucleic acid sequence encompassed bySEQ ID NO: 40 except that one or more nucleotides from the 5′ and/or 3′end have been deleted. Preferably, a subsequence contains at least 1020nucleotides, more preferably at least 1080 nucleotides, and mostpreferably at least 1140 nucleotides. A fragment of SEQ ID NO: 41 is apolypeptide having one or more amino acids deleted from the amino and/orcarboxy terminus of this amino acid sequence. Preferably, a fragmentcontains at least 340 amino acid residues, more preferably at least 360amino acid residues, and most preferably at least 380 amino acidresidues.

A subsequence of SEQ ID NO: 42 is a nucleic acid sequence encompassed bySEQ ID NO: 42 except that one or more nucleotides from the 5′ and/or 3′end have been deleted. Preferably, a subsequence contains at least 765nucleotides, more preferably at least 810 nucleotides, and mostpreferably at least 855 nucleotides. A fragment of SEQ ID NO: 43 is apolypeptide having one or more amino acids deleted from the amino and/orcarboxy terminus of this amino acid sequence. Preferably, a fragmentcontains at least 255 amino acid residues, more preferably at least 270amino acid residues, and most preferably at least 285 amino acidresidues.

A subsequence of SEQ ID NO: 44 is a nucleic acid sequence encompassed bySEQ ID NO: 44 except that one or more nucleotides from the 5′ and/or 3′end have been deleted. Preferably, a subsequence contains at least 1110nucleotides, more preferably at least 1200 nucleotides, and mostpreferably at least 1290 nucleotides. A fragment of SEQ ID NO: 45 is apolypeptide having one or more amino acids deleted from the amino and/orcarboxy terminus of this amino acid sequence. Preferably, a fragmentcontains at least 370 amino acid residues, more preferably at least 400amino acid residues, and most preferably at least 430 amino acidresidues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. The allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

In a second embodiment, the present invention relates to isolatednucleic acid sequences which have a degree of homology to SEQ ID NO: 40of at least about 75%, preferably at least about 80%, more preferably atleast about 85%, even more preferably at least about 90%, mostpreferably at least about 95%, and even most preferably at least about97%.

In another second embodiment, the present invention relates to isolatednucleic acid sequences which have a degree of homology to SEQ ID NO: 42of at least about 90%, preferably at least about 95%, and morepreferably at least about 97%.

In another second embodiment, the present invention relates to isolatednucleic acid sequences which have a degree of homology to SEQ ID NO: 44of at least about 75%, preferably at least about 80%, more preferably atleast about 85%, even more preferably at least about 90%, mostpreferably at least about 95%, and even most preferably at least about97%.

For purposes of the present invention, the degree of homology betweentwo nucleic acid sequences is determined by the Vector NTI AlignXsoftware package (Informax Inc., Bethesda, Md.) using the followingdefaults: pairwise alignment, gap opening penalty of 15, gap extensionpenalty of 6.6, and score matrix: swgapdnamt.

In a third embodiment, the present invention relates to isolated nucleicacid sequences encoding polypeptides having UDP-glucose 6-dehydrogenase,UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosaminepyrophosphorylase activity, which hybridize under very low stringencyconditions, preferably low stringency conditions, more preferably mediumstringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the nucleic acidsequence of SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44, (ii) thecDNA sequence contained in SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO:44, or (iii) a complementary strand of (i) or (ii) (J. Sambrook, E. F.Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, N.Y.). The subsequence of SEQ ID NO: 40,SEQ ID NO: 42, or SEQ ID NO: 44 may be at least 100 nucleotides orpreferably at least 200 nucleotides. Moreover, the respectivesubsequence may encode a polypeptide fragment which has UDP-glucose6-dehydrogenase, UDP-glucose pyrophosphorylase, orUDP-N-acetylglucosamine pyrophosphorylase activity.

The nucleic acid sequence of SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO:44, or subsequences thereof, as well as the amino acid sequence of SEQID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, or a fragment thereof, maybe used to design nucleic acid probes to identify and clone DNA encodingpolypeptides having UDP-glucose 6-dehydrogenase, UDP-glucosepyrophosphorylase, or UDP-N-acetylglucosamine pyrophosphorylaseactivity, respectively, from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 15, preferably at least 25, and more preferably at least 35nucleotides in length. Longer probes can also be used. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA which hybridizes with the probes described aboveand which encodes a polypeptide having UDP-glucose 6-dehydrogenase,UDP-glucose pyrophosphorylase, or UDP-N-acetylglucosaminepyrophosphorylase activity. Genomic or other DNA from such otherorganisms may be separated by agarose or polyacrylamide gelelectrophoresis, or other separation techniques. DNA from the librariesor the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA which is homologous with SEQ ID NO: 40, SEQ ID NO: 42, orSEQ ID NO: 44, or a subsequence thereof, the carrier material is used ina Southern blot. For purposes of the present invention, hybridizationindicates that the nucleic acid sequence hybridizes to a labeled nucleicacid probe corresponding to the nucleic acid sequence shown in SEQ IDNO: 40, SEQ ID NO: 42, or SEQ ID NO: 44, its complementary strand, or asubsequence thereof, under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions are detected using X-ray film.

In a preferred embodiment, the nucleic acid probe is a nucleic acidsequence which encodes the polypeptide of SEQ ID NO: 41, SEQ ID NO: 43,or SEQ ID NO: 45; or a subsequence thereof. In another preferredembodiment, the nucleic acid probe is SEQ ID NO: 40, SEQ ID NO: 42, orSEQ ID NO: 44. In another preferred embodiment, the nucleic acid probeis the nucleic acid sequence contained in plasmid pMRT106 which iscontained in Escherichia coli NRRL B-30536, wherein the nucleic acidsequence encodes polypeptides having UDP-glucose 6-dehydrogenase,UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosaminepyrophosphorylase activity.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at 5° C. to 10° C. belowthe calculated T_(n), using the calculation according to Bolton andMcCarthy (1962, Proceedings of the National Academy of Sciences USA48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasicphosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standardSouthern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a fourth embodiment, the present invention relates to isolatednucleic acid sequences which encode variants of the polypeptide havingan amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45comprising a substitution, deletion, and/or insertion of one or moreamino acids.

The amino acid sequences of the variant polypeptides may differ from theamino acid sequence of SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45,by an insertion or deletion of one or more amino acid residues and/orthe substitution of one or more amino acid residues by different aminoacid residues. Preferably, amino acid changes are of a minor nature,that is conservative amino acid substitutions that do not significantlyaffect the folding and/or activity of the protein; small deletions,typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

Modification of a nucleic acid sequence of the present invention may benecessary for the synthesis of polypeptides substantially similar to thepolypeptide. The term “substantially similar” to the polypeptide refersto non-naturally occurring forms of the polypeptide. These polypeptidesmay differ in some engineered way from the polypeptide isolated from itsnative source, e.g., variants that differ in specific activity,thermostability, pH optimum, or the like. The variant sequence may beconstructed on the basis of the nucleic acid sequence presented as thepolypeptide encoding part of SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO:44, e.g., a subsequence thereof, and/or by introduction of nucleotidesubstitutions which do not give rise to another amino acid sequence ofthe polypeptide encoded by the nucleic acid sequence, but whichcorresponds to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the isolated nucleic acidsequence of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In thelatter technique, mutations are introduced at every positively chargedresidue in the molecule, and the resultant mutant molecules are testedfor enzyme activity to identify amino acid residues that are critical tothe activity of the molecule. Sites of substrate-enzyme interaction canalso be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The polypeptides encoded by the isolated nucleic acid sequences of thepresent invention have at least 20%, preferably at least 40%, morepreferably at least 60%, even more preferably at least 80%, even morepreferably at least 90%, and most preferably at least 100% of theUDP-glucose 6-dehydrogenase activity of the polypeptide of SEQ ID NO:41, the UDP-glucose pyrophosphorylase activity of the polypeptide of SEQID NO: 43, or the UDP-N-acetylglucosamine pyrophosphorylase activity ofthe polypeptide of SEQ ID NO: 45.

The nucleic acid sequences of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by the nucleic acid sequence isproduced by the source or by a cell in which the nucleic acid sequencefrom the source has been inserted. In a preferred embodiment, thepolypeptide encoded by a nucleic acid sequence of the present inventionis secreted extracellularly.

The nucleic acid sequences may be obtained from a bacterial source. Forexample, these polypeptides may be obtained from a gram positivebacterium such as a Bacillus strain, e.g., Bacillus agaradherens,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis; or a Streptomyces strain, e.g., Streptomyces lividans orStreptomyces murinus; or from a gram negative bacterium, e.g., E. colior Pseudomonas sp.

In a preferred embodiment, the nucleic acid sequences are obtained froma Streptococcus or Pastuerella strain.

In a more preferred embodiment, the nucleic acid sequences are obtainedfrom a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcusuberis, or Streptococcus equi subs. zooepidemicus strain, or aPasteurella multocida strain.

In a most preferred embodiment, the nucleic acid sequences are obtainedfrom Streptococcus equisimilis, e.g., the nucleic acid sequence setforth in SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44. In another mostpreferred embodiment, the nucleic acid sequence is the sequencecontained in plasmid pMRT106 which is contained in Escherichia coli NRRLB-30536. In further most preferred embodiment, the nucleic acid sequenceis SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such nucleic acid sequences may be identified and obtainedfrom other sources including microorganisms isolated from nature (e.g.,soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms from natural habitats are wellknown in the art. The nucleic acid sequence may then be derived bysimilarly screening a genomic or cDNA library of another microorganism.Once a nucleic acid sequence encoding a polypeptide has been detectedwith the probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

The present invention also relates to mutant nucleic acid sequencescomprising at least one mutation in the polypeptide coding sequence ofSEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, in which the mutantnucleic acid sequence encodes a polypeptide which consists of SEQ ID NO:42, SEQ ID NO: 43, and SEQ ID NO: 45, respectively.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Streptococcus, or another orrelated organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleic acid sequence.

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals.

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides.

The present invention also relates to methods for producing apolypeptide having UDP-N-acetylglucosamine pyrophosphorylase activitycomprising (a) cultivating a host cell under conditions suitable forproduction of the polypeptide; and (b) recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, and small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989).

The present invention further relates to the isolated polypeptideshaving UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, orUDP-N-acetylglucosamine pyrophosphorylase activity encoded by thenucleic acid sequences described above.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Primers and Oligos

All primers and oligos were purchased (MWG Biotech Inc., High Point,N.C.)

Example 1 PCR Amplification and Cloning of the Streptococcus equisimilishasA Gene and the Bacillus subtilis tuaD, gtaB, and gcaD Genes

The Streptococcus equisimilis hyaluronan synthase gene (hasA, accessionnumber AF023876, SEQ ID NOs: 1 [DNA sequence] and 2 [deduced amino acidsequence]) was PCR amplified from plasmid pKKseD (Weigel, 1997, Journalof Biological Chemistry 272: 32539-32546) using primers 1 and 2:

Primer 1: (SEQ ID NO: 3)5′-GAGCTCTATAAAAATGAGGAGGGAACCGAATGAGAACATTAAAAAAC CT-3′ Primer 2:(SEQ ID NO: 4) 5′-GTTAACGAATTCAGCTATGTAGGTACCTTATAATAATTTTTTACGTG T-3′

PCR amplifications were conducted in triplicate in 50 μl reactionscomposed of 1 ng of pKKseD DNA, 0.4 μM each of primers 1 and 2, 200 μMeach of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II (Applied Biosystems,Inc., Foster City, Calif.) with 2.5 mM MgCl₂, and 2.5 units of AmpliTaqGold™ DNA polymerase (Applied Biosystems, Inc., Foster City, Calif.).The reactions were performed in a RoboCycler 40 thermacycler(Stratagene, Inc., La Jolla, Calif.) programmed for 1 cycle at 95° C.for 9 minutes; 3 cycles each at 95° C. for 1 minute, 52° C. for 1minute, and 72° C. for 1 minute; 27 cycles each at 95° C. for 1 minute,55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for5 minutes. The PCR product was visualized using a 0.8% agarose gel with44 mM Tris Base, 44 mM boric acid, 0.5 mM EDTA buffer (0.5×TBE). Theexpected fragment was approximately 1200 bp.

The 1200 bp PCR fragment was cloned into pCR2.1 using the TA-TOPOCloning Kit (Stratagene, Inc., La Jolla, Calif.) and transformed into E.coli OneShot™ competent cells according to the manufacturers'instructions (Stratagene, Inc., La Jolla, Calif.). Transformants wereselected at 37° C. after 16 hours of growth on 2× yeast-tryptone (YT)agar plates supplemented with 100 μg of ampicillin per ml. Plasmid DNAfrom these transformants was purified using a QIAGEN robot (QIAGEN,Valencia, Calif.) according to the manufacturer's instructions and theDNA sequence of the inserts confirmed by DNA sequencing using M13 (−20)forward and M13 reverse primers (Invitrogen, Inc, Carlsbad, Calif.) andthe following internal primers. The plasmid harboring the 1200 bp PCRfragment was designated pCR2.1-sehasA (FIG. 3).

Primer 3: 5′-GTTGACGATGGAAGTGCTGA-3′ (SEQ ID NO: 5) Primer 4:5′-ATCCGTTACAGGTAATATCC-3′ (SEQ ID NO: 6) Primer 5:5′-TCCTTTTGTAGCCCTATGGA-3′ (SEQ ID NO: 7) Primer 6:5′-TCAGCACTTCCATCGTCAAC-3′ (SEQ ID NO: 8) Primer 7:5′-GGATATTACCTGTAACGGAT-3′ (SEQ ID NO: 9) Primer 8:5′-TCCATAGGGCTACAAAAGGA-3′ (SEQ ID NO: 10)

The Bacillus subtilis UDP-glucose-6-dehydrogenase gene (tuaD, accessionnumber BG12691, SEQ ID NOs: 11 [DNA sequence] and 12 [deduced amino acidsequence]) was PCR amplified from Bacillus subtilis 168 (BGSC 1A1,Bacillus Genetic Stock Center, Columbus, Ohio) using primers 9 and 10:

Primer 9: (SEQ ID NO: 13) 5′-GGTACCGACACTGCGACCATTATAAA-3′ Primer 10:(SEQ ID NO: 14) 5′-GTTAACGAATTCCAGCTATGTATCTAGACAGCTTCAACCAAGTAACA CT-3′

PCR amplifications were carried out in triplicate in 30 μl reactionscomposed of 50 ng of Bacillus subtilis 168 chromosomal DNA, 0.3 μM eachof primers 9 and 10, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCRBuffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaq Gold™ DNApolymerase. The reactions were performed in a RoboCycler 40 programmedfor 1 cycle at 95° C. for 9 minutes; 5 cycles each at 95° C. for 1minute, 50° C. for 1 minute, and 72° C. for 1.5 minutes; 32 cycles eachat 95° C. for 1 minute, 54° C. for 1 minute, and 72° C. for 1.5 minute;and 1 cycle at 72° C. for 7 minutes. The PCR product was visualized in a0.8% agarose gel using 0.5×TBE buffer. The expected fragment wasapproximately 1400 bp.

The 1400 bp PCR fragment was cloned into pCR2.1 using the TA-TOPOCloning Kit and transformed into E. coli OneShot™ competent cellsaccording to the manufacturers' instructions. Plasmid DNA was purifiedusing a QIAGEN robot according to the manufacturer's instructions andthe DNA sequence of the inserts confirmed by DNA sequencing using M13(−20) forward and M13 reverse primers and the following internalprimers. The plasmid harboring the 1400 bp PCR fragment was designatedpCR2.1-tuaD (FIG. 4).

Primer 11: (SEQ ID NO: 15) 5′-AGCATCTTAACGGCTACAAA-3′ Primer 12:(SEQ ID NO: 16) 5′-TGTGAGCGAGTCGGCGCAGA-3′ Primer 13: (SEQ ID NO: 17)5′-GGGCGCCCATGTAAAAGCAT-3′ Primer 14: (SEQ ID NO: 18)5′-TTTGTAGCCGTTAAGATGCT-3′ Primer 15: (SEQ ID NO: 19)5′-TCTGCGCCGACTCGCTCACA-3′ Primer 16: (SEQ ID NO: 20)5′-ATGCTTTTACATGGGCGCCC-3′

The Bacillus subtilis UTP-glucose-1-phosphate uridylyltransferase gene(gtaB, accession number BG10402, SEQ ID NOs: 21 [DNA sequence] and 22[deduced amino acid sequence]) was PCR amplified from Bacillus subtilis168 using primers 17 and 18:

Primer 17: (SEQ ID NO: 23) 5′-TCTAGATTTTTCGATCATAAGGAAGGT-3′ Primer 18:(SEQ ID NO: 24) 5′-GTTAACGAATTCCAGCTATGTAGGATCCAATGTCCAATAGC CTTTTTGT-3′

PCR amplifications were carried out in triplicate in 30 μl reactionscomposed of 50 ng of Bacillus subtilis 168 chromosomal DNA, 0.3 μM eachof primers 17 and 18, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCRBuffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaq Gold™ DNApolymerase. The reactions were performed in a RoboCycler 40 programmedfor 1 cycle at 95° C. for 9 minutes; 5 cycles each at 95° C. for 1minute, 50° C. for 1 minute, and 72° C. for 1.5 minutes; 32 cycles eachat 95° C. for 1 minute, 54° C. for 1 minute, and 72° C. for 1.5 minute;and 1 cycle at 72° C. for 7 minutes. The PCR product was visualized in a0.8% agarose-0.5×TBE gel. The expected fragment was approximately 900bp.

The 900 bp PCR fragment was cloned into pCR2.1 using the TA-TOPO cloningkit and transformed into E. coli OneShot™ competent cells according tothe manufacturer's instructions. Plasmid DNA was purified using a QIAGENrobot according to the manufacturer's instructions and the DNA sequenceof the inserts confirmed by DNA sequencing using M13 (−20) forward andM13 reverse primers and the following internal primers. The plasmidharboring the 900 bp PCR fragment was designated pCR2.1-gtaB (FIG. 5).

Primer 19: (SEQ ID NO: 25) 5′-AAAAAGGCTTCTAACCTGGC-3′ Primer 20:(SEQ ID NO: 26) 5′-AAACCGCCTAAAGGCACAGC-3′ Primer 21: (SEQ ID NO: 27)5′-GCCAGGTTAGAAGCCTTTTT-3′ Primer 22: (SEQ ID NO: 28)5′-GCTGTGCCTTTAGGCGGTTT-3′

The Bacillus subtilis UDP-N-acetylglucosamine pyrophosphorylase gene(gcaD, accession number BG10113, SEQ ID NOs: 29 [DNA sequence] and 30[deduced amino acid sequence]) was PCR amplified from Bacillus subtilis168 using primers 23 and 24:

Primer 23: (SEQ ID NO: 31) 5′-GGATCCTTTCTATGGATAAAAGGGAT-3′ Primer 24:(SEQ ID NO: 32) 5′-GTTAACAGGATTATTTTTTATGAATATTTTT-3′

PCR amplifications were carried out in triplicate in 30 μl reactionscomposed of 50 ng of Bacillus subtilis 168 chromosomal DNA, 0.3 μM eachof primers 23 and 24, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCRBuffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaq Gold™ DNApolymerase. The reactions were performed in a RoboCycler 40 programmedfor 1 cycle at 95° C. for 9 minutes; 5 cycles each at 95° C. for 1minute, 50° C. for 1 minute, and 72° C. for 1.5 minutes; 32 cycles eachat 95° C. for 1 minute, 54° C. for 1 minute, and 72° C. for 1.5 minute;and 1 cycle at 72° C. for 7 minutes. The PCR product was visualized in a0.8% agarose-0.5×TBE gel. The expected fragment was approximately 1500bp.

The 1500 bp PCR fragment was cloned into pCR2.1 using the TA-TOPOcloning kit and transformed into E. coli OneShot™ competent cellsaccording to the manufacturer's instructions. Plasmid DNA was purifiedusing a QIAGEN robot according to the manufacturer's instructions andthe DNA sequence of the inserts confirmed by DNA sequencing using M13(−20) forward and M13 reverse primers and the following internalprimers. The plasmid harboring the 900 bp PCR fragment was designatedpCR2.1-gcaD (FIG. 6).

Primer 25: (SEQ ID NO: 33) 5′-CAGAGACGATGGAACAGATG-3′ Primer 26:(SEQ ID NO: 34) 5′-GGAGTTAATGATAGAGTTGC-3′ Primer 27: (SEQ ID NO: 35)5′-GAAGATCGGGAATTTTGTAG-3′ Primer 28: (SEQ ID NO: 36)5′-CATCTGTTCCATCGTCTCTG-3′ Primer 29: (SEQ ID NO: 37)5′-GCAACTCTATCATTAACTCC-3′ Primer 30: (SEQ ID NO: 38)5′-CTACAAAATTCCCGATCTTC-3′

Example 2 Construction of the hasA/tuaD/gtaB Operon

Plasmids pDG268Δneo-cryIIIAstab/Sav (U.S. Pat. No. 5,955,310) andpCR2.1-tuaD (Example 1, FIG. 4) were digested with KpnI and HpaI. Thedigestions were resolved on a 0.8% agarose gel using 0.5×TBE buffer andthe larger vector fragment (approximately 7700 bp) frompDG268Δneo-cryIIIAstab/Sav and the smaller tuaD fragment (approximately1500 bp) from pCR2.1-tuaD were gel-purified using the QIAquick DNAExtraction kit according to the manufacturer's instructions (QIAGEN,Valencia, Calif.). The two purified fragments were ligated together withT4 DNA ligase according to the manufacturer's instructions (RocheApplied Science; Indianapolis, Ind.) and the ligation mix wastransformed into E. coli SURE competent cells (Stratagene, Inc., LaJolla, Calif.). Transformants were selected on 2×YT agar platessupplemented with 100 mg of ampicillin per ml.

Plasmid DNA was purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by KpnI plusHpaI digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correctplasmid was identified by the presence of an approximately 1500 bpKpnI/HpaI tuaD fragment and was designated pHA1 (FIG. 7).

Plasmids pHA1 and pCR2.1-gtaB (Example 1, FIG. 5) were digested withXbaI and HpaI. The digestions were resolved on a 0.8% agarose gel using0.5×TBE buffer and the larger vector fragment from pHA1 (approximately9200 bp) and the smaller gtaB fragment (approximately 900 bp) frompCR2.1-gtaB were gel-purified from a 0.8% agarose-0.5×TBE buffer gelusing the QIAquick DNA Extraction Kit according to the manufacturer'sinstructions. These two purified fragments were ligated together with T4DNA ligase and the ligation mix was used to transform E. coli SUREcompetent cells. Transformants were selected on 2×YT agar platessupplemented with 100 mg of ampicillin per ml at 37° C.

Plasmids were purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by XbaI plusHpaI digestion. The digestions were resolved on a 0.8% agarose-0.5×TBEbuffer gel. The correct plasmid was identified by the presence of anapproximately 900 bp XbaI/HpaI gtaB fragment and was designated pHA2(FIG. 8).

Plasmids pHA2 and pCR2.1-sehasA (Example 1, FIG. 3) were digested withSacI plus KpnI. The digestions were resolved on a 0.8% agarose-0.5×TBEbuffer gel. The larger vector fragment (approximately 10000 bp) frompHA2 and the smaller hasA fragment (approximately 1300 bp) frompCR2.1-sehasA were gel-purified from a 0.8% agarose-0.5×TBE buffer gelusing the QIAquick DNA Extraction kit according to the manufacturer'sinstructions. The two purified fragments were ligated together with T4DNA ligase and the ligation mix was used to transform E. coli SUREcompetent cells. Transformants were selected on 2×YT agar platessupplemented with 100 μg of ampicillin per ml at 37° C. Plasmids werepurified from several transformants using a QIAGEN robot according tothe manufacturer's instructions and analyzed by SacI plus KpnIdigestion. The digestions were resolved on a 0.8% agarose-0.5×TBE buffergel. The correct plasmid was identified by the presence of anapproximately 1300 bp SacI/KpnI hasA fragment and was designated pHA3(FIG. 9).

Example 3 Construction of the hasA/tuaD/gtaB/gcaD Operon

Plasmids pHA2 (Example 2, FIG. 8) and pCR2.1-gcaD (Example 1, FIG. 6)were digested with BamHI and HpaI. The digestions were resolved on a0.8% agarose gel using 0.5×TBE buffer and the larger vector fragment(approximately 10,000 bp) from pHA2 and the smaller gcaD fragment(approximately 1,400 bp) from pCR2.1-gcaD were gel-purified from a 0.8%agarose-0.5×TBE buffer gel using the QIAquick DNA Extraction Kitaccording to the manufacturer's instructions. These two purifiedfragments were ligated together with T4 DNA ligase and the ligation mixwas used to transform E. coli SURE competent cells. Transformants wereselected on 2×YT agar plates supplemented with 100 μg of ampicillin perml at 37° C.

Plasmids were purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by XbaI plusHpaI digestion. The digestions were resolved on a 0.8% agarose-0.5×TBEbuffer gel. The correct plasmid was identified by the presence of anapproximately 1400 bp BamHI/HpaI gcaD fragment and was designated pHA4(FIG. 10).

Plasmids pHA4 and pCR2.1-sehasA (Example 1, FIG. 3) were digested withSacI and KpnI. The digestions were resolved on a 0.8% agarose-0.5×TBEbuffer gel. The larger vector fragment (approximately 11,000 bp) frompHA4 and the smaller hasA fragment (approximately 1,300 bp) frompCR2.1-sehasA were gel-purified from a 0.8% agarose-0.5×TBE buffer gelusing the QIAquick DNA Extraction kit according to the manufacturer'sinstructions. The two purified fragments were ligated together with T4DNA ligase and the ligation mix was used to transform E. coli SUREcompetent cells. Transformants were selected on 2×YT agar platessupplemented with 100 μg of ampicillin per ml at 37° C. Plasmids werepurified from several transformants using a QIAGEN robot according tothe manufacturer's instructions and analyzed by SacI plus KpnIdigestion. The digestions were resolved on a 0.8% agarose-0.5×TBE buffergel. The correct plasmid was identified by the presence of anapproximately 1,300 bp SacI/KpnI hasA fragment and was designated pHA5(FIG. 11).

Example 4 Construction of the hasA/tuaD/gcaD Operon

Plasmids pHA1 (Example 2, FIG. 7) and pCR2.1-gcaD (Example 1, FIG. 6)were digested with BamHI and HpaI. The digestions were resolved on a0.8% agarose gel using 0.5×TBE buffer and the larger vector fragmentfrom pHA1 (approximately 9,200 bp) and the smaller gcaD fragment(approximately 1400 bp) from pCR2.1-gcaD were gel-purified from a 0.8%agarose-0.5×TBE buffer gel using the QIAquick DNA Extraction Kitaccording to the manufacturer's instructions. These two purifiedfragments were ligated together with T4 DNA ligase and the ligation mixwas used to transform E. coli SURE competent cells. Transformants wereselected on 2×YT agar plates supplemented with 100 μg of ampicillin perml at 37° C.

Plasmids were purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by BamHI plusHpaI digestion. The digestions were resolved on a 0.8% agarose-0.5×TBEbuffer gel. The correct plasmid was identified by the presence of anapproximately 1400 bp BamHI/HpaI gtaB fragment and was designated pHA6(FIG. 12).

Plasmids pHA6 and pCR2.1-sehasA (Example 1, FIG. 3) were digested withSacI plus KpnI. The digestions were resolved on a 0.8% agarose-0.5×TBEbuffer gel. The larger vector fragment (approximately 10,200 bp) frompHA6 and the smaller hasA fragment (approximately 1,300 bp) frompCR2.1-sehasA were gel-purified from a 0.8% agarose-0.5×TBE buffer gelusing the QIAquick DNA Extraction kit according to the manufacturer'sinstructions. The two purified fragments were ligated together with T4DNA ligase and the ligation mix was used to transform E. coli SUREcompetent cells. Transformants were selected on 2×YT agar platessupplemented with 100 μg of ampicillin per ml. Plasmids were purifiedfrom several transformants using a QIAGEN robot according to themanufacturer's instructions and analyzed by SacI plus KpnI digestion.The digestions were resolved on a 0.8% agarose-0.5×TBE buffer gel. Thecorrect plasmid was identified by the presence of an approximately 1300bp SacI/KpnI hasA fragment and was designated pHA7 (FIG. 13).

Example 5 Construction of Bacillus subtilis RB161

Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digestedwith SacI. The digested plasmid was then purified using a QIAguick DNAPurification Kit according to the manufacturer's instructions, andfinally digested with NotI. The largest plasmid fragment ofapproximately 6800 bp was gel-purified using a QIAquick DNA GelExtraction Kit from a 0.8% agarose-0.5×TBE gel according to themanufacturer's instructions (QIAGEN, Valencia, Calif.). The recoveredvector DNA was then ligated with the DNA insert described below.

Plasmid pHA3 (Example 2, FIG. 9) was digested with SacI. The digestedplasmid was then purified as described above, and finally digested withNotI. The smallest plasmid fragment of approximately 3800 bp wasgel-purified as described above. The recovered vector and DNA insertwere ligated using the Rapid DNA Cloning Kit (Roche Applied Science;Indianapolis, Ind.) according to the manufacturer's instructions. Priorto transformation in Bacillus subtilis, the ligation described above waslinearized using ScaI to ensure double cross-over integration in thechromosome rather than single cross-over integration in the chromosome.Competent cells of Bacillus subtilis 168Δ4 were transformed with theligation products digested with ScaI. Bacillus subtilis 168Δ4 is derivedfrom the Bacillus subtilis type strain 168 (BGSC 1A1, Bacillus GeneticStock Center, Columbus, Ohio) and has deletions in the spollAC, aprE,nprE, and amyE genes. The deletion of these four genes was performedessentially as described for Bacillus subtilis A164Δ5, which isdescribed in detail in U.S. Pat. No. 5,891,701.

Bacillus subtilis chloramphenicol-resistant transformants were selectedat 34° C. after 16 hours of growth on Tryptose blood agar base (TBAB)plates supplemented with 5 μg of chloramphenicol per ml. To screen forintegration of the plasmid by double cross-over at the amyE locus,Bacillus subtilis primary transformants were patched on TBAB platessupplemented with 6 μg of neomycin per ml and on TBAB platessupplemented with 5 μg of chloramphenicol per ml. Integration of theplasmid by double cross-over at the amyE locus does not incorporate theneomycin resistance gene and therefore renders the strain neomycinsensitive. Isolates were also patched onto minimal plates to visualizewhether or not these were producing hyaluronic acid. Hyaluronic acidproducing isolates have a “wet” phenotype on minimal plates. Using thisplate screen, chloramphenicol resistant and neomycin sensitive “wet”transformants (due to hyaluronic acid production) were isolated at 37°C.

Genomic DNA was isolated from the “wet”, chloramphenicol resistant, andneomycin sensitive Bacillus subtilis 168Δ4 transformants using a QIAGENtip-20 column (QIAGEN, Valencia, Calif.) according to the manufacturer'sinstructions. PCR amplifications were performed on these transformantsusing the synthetic oligonucleotides below, which are based on the hasA,tuaD, and gtaB gene sequences, to confirm the presence and integrity ofthese genes in the operon of the Bacillus subtilis transformants.

The amplification reactions (25 μl) were composed of approximately 50 ngof genomic DNA of the Bacillus subtilis 168Δ4 transformants, 0.5 μM ofeach primer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II,3 mM MgCl₂, and 0.625 units of AmpliTaq Gold™ DNA polymerase. Thereactions were incubated in a RoboCycler 40 Temperature Cyclerprogrammed for one cycle at 95° C. for 9 minutes; 30 cycles each at 95°C. for 1 minute, 55° C. for 1 minute, and 72° C. for 2 minutes; and afinal cycle at 72° C. for 7 minutes.

Primers 3 and 8 were used to confirm the presence of the hasA gene,primers 3 and 16 to confirm the presence of the tuaD gene, and primers 3and 22 to confirm the presence of the gtaB gene. The Bacillus subtilis168Δ4 hasA/tuaD/gtaB integrant was designated Bacillus subtilis RB158.

Genomic DNA was isolated from Bacillus subtilis RB158 using a QIAGENtip-20 column according to the manufacturer's instructions, and was usedto transform competent Bacillus subtilis A164Δ5 (deleted at the spollAC,aprE, nprE, amyE, and srfC genes; see U.S. Pat. No. 5,891,701).Transformants were selected on TBAB plates supplemented with 5 μg ofchloramphenicol per ml at 37° C. A Bacillus subtilis A164Δ5hasA/tuaD/gtaB integrant was identified by its “wet” phenotype anddesignated Bacillus subtilis RB161.

Example 6 Construction of Bacillus subtilis RB163

Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digestedwith SacI. The digested plasmid was then purified using a QIAquick DNAPurification Kit according to the manufacturer's instructions, andfinally digested with NotI. The largest plasmid fragment ofapproximately 6,800 bp was gel-purified using a QIAquick DNA GelExtraction Kit from a 0.8% agarose-0.5×TBE gel according to themanufacturer's instructions. The recovered vector DNA was then ligatedwith the DNA insert described below.

Plasmid pHA7 (Example 4, FIG. 13) was digested with SacI. The digestedplasmid was then purified as described above, and finally digested withNotI. The smallest plasmid fragment of approximately 4,300 bp wasgel-purified as described above. The recovered vector and DNA insertwere ligated using the Rapid DNA Cloning Kit according to themanufacturer's instructions. Prior to transformation in Bacillussubtilis, the ligation described above was linearized using ScaI toensure double cross-over integration in the chromosome rather thansingle cross-over integration in the chromosome. Bacillus subtilis 168Δ4competent cells were transformed with the ligation digested with therestriction enzyme ScaI.

Bacillus subtilis chloramphenicol-resistant transformants were selectedon TBAB plates supplemented with 5 μg of chloramphenicol per ml at 37°C. To screen for integration of the plasmid by double cross-over at theamyE locus, Bacillus subtilis primary transformants were patched on TBABplates supplemented with 6 μg of neomycin per ml and on TBAB platessupplemented with 5 μg of chloramphenicol per ml to isolatechloramphenicol resistant and neomycin sensitive “wet” transformants(due to hyaluronic acid production).

Genomic DNA was isolated from the “wet”, chloramphenicol resistant, andneomycin sensitive Bacillus subtilis 168Δ4 transformants using a QIAGENtip-20 column according to the manufacturer's instructions. PCRamplifications were performed on these transformants using primers 3, 8,16, 22 and primer 30 (Example 1) to confirm the presence and integrityof these genes in the operon of the Bacillus subtilis transformants. Theamplification reactions (25 μl) were composed of approximately 50 ng ofgenomic DNA of the Bacillus subtilis 168Δ4 transformants, 0.5 μM of eachprimer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR buffer, 3 mMMgCl₂, and 0.625 units of AmpliTaq Gold™ DNA polymerase. The reactionswere incubated in a RoboCycler 40 Temperature Cycler programmed for onecycle at 95° C. for 9 minutes; 30 cycles each at 95° C. for 1 minute,55° C. for 1 minute, and 72° C. for 2 minutes; and a final cycle at 72°C. for 7 minutes.

Primers 3 and 8 were used to confirm the presence of the hasA gene,primers 3 and 16 to confirm the presence of the tuaD gene, primers 3 and22 to confirm the presence of the gtaB gene, and primers 3 and 30 toconfirm the presence of the gcaD gene. The Bacillus subtilis 168Δ4hasA/tuaD/gcaD integrant was designated Bacillus subtilis RB160.

Genomic DNA was isolated from Bacillus subtilis RB160 using a QIAGENtip-20 column according to the manufacturer's instructions, and was usedto transform competent Bacillus subtilis A164Δ5. Transformants wereselected on TBAB plates containing 5 μg of chloramphenicol per ml, andgrown at 37° C. for 16 hours. The Bacillus subtilis A164Δ5hasA/tuaD/gcaD integrant was identified by its “wet” phenotype anddesignated Bacillus subtilis RB163.

Example 7 Construction of Bacillus subtilis TH-1

The hyaluronan synthase (has) operon was obtained from Streptococcusequisimilis using the following procedure. The has operon is composed ofthe hasA, hasB, hasC, and hasD genes. Approximately 20 μg ofStreptococcus equisimilis D181 (Kumari and Weigel, 1997, Journal ofBiological Chemistry 272: 32539-32546) chromosomal DNA was digested withHindIII and resolved on a 0.8% agarose-0.5×TBE gel. DNA in the 3-6 kbrange was excised from the gel and purified using the QIAquick DNA GelExtraction Kit according to the manufacturer's instructions. Therecovered DNA insert was then ligated with the vector DNA describedbelow.

Plasmid pUC18 (2 μg) was digested with HindIII and the 5′ protrudingends were dephosphorylated with shrimp alkaline phosphatase according tothe manufacturer's instructions (Roche Applied Science; Indianapolis,Ind.). The dephosphorylated vector and DNA insert were ligated using theRapid DNA Cloning Kit according to the manufacturer's instructions. Theligation was used to transform E. coli XL10 Gold Kan competent cells(Stratagene, Inc., La Jolla, Calif.). Cells were plated onto Luria brothplates (100 μg/ml ampicillin) and incubated overnight at 37° C. Fiveplates containing approximately 500 colonies/plate were probed witholigo 952-55-1, shown below, which is a 54 bp sequence identical to thecoding strand near the 3′ end of the Streptococcus equisimilis D181 hasAgene (nucleotides 1098-1151 with respect to the A residue of the ATGtranslation start codon).

Primer 31: (SEQ ID NO: 39)5′-GTGTCGGAACATTCATTACATGCTTAAGCACCCGCTGTCCTTCTT GTTATCTCC-3′

The oligonucleotide probe was DIG-labeled using the DIG Oligonucleotide3′-end Labeling Kit according to the manufacturer's instructions (RocheApplied Science; Indianapolis, Ind.). Colony hybridization andchemiluminescent detection were performed as described in “THE DIGSYSTEM USER'S GUIDE FOR FILTER HYBRIDIZATION”, Boehringer Mannheim GmbH.

Seven colonies were identified that hybridized to the probe. Plasmid DNAfrom one of these transformants was purified using a QIAGEN robot(QIAGEN, Valencia, Calif.) according to the manufacturer's instructions,digested with HindIII, and resolved on a 0.8% agarose gel using 0.5×TBEbuffer. The DNA insert was shown to be approximately 5 kb in size. Thisplasmid was designated pMRT106 (FIG. 14).

The DNA sequence of the cloned fragment was determined using the EZ::TN™<TET-1> Insertion Kit according to the manufacturer's instructions(Epicenter Technologies, Madison, Wis.). The sequencing revealed thatthe cloned DNA insert contained the last 1156 bp of the Streptococcusequisimilis hasA gene followed by three other genes designated hasB,hasC, and hasD; presumably all four genes are contained within a singleoperon and are therefore co-transcribed. The Streptococcus equisimilishasB gene is contained in nucleotides 1411-2613 (SEQ ID NOs: 40 [DNAsequence] and 41 [deduced amino acid sequence]) of the fragment, andStreptococcus equisimilis hasC gene in nucleotides 2666-3565 (SEQ IDNOs: 42 [DNA sequence] and 43 [deduced amino acid sequence]) of thefragment, and Streptococcus equisimilis hasD gene in nucleotides3735-5114 (SEQ ID NOs: 44 [DNA sequence] and 45 [deduced amino acidsequence]) of the fragment.

The polypeptides encoded by the Streptococcus equisimilis hasB and hasCgenes show some homology to those encoded by the hasB and hasC genes,respectively, from the Streptococcus pyogenes has operon sequence(Ferretti et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98 (8),4658-4663). The degree of identity was determined by the Clustal method(Higgins, 1989, CABIOS 5: 151-153) using the Vector NTI AlignX software(Informax Inc., Bethesda, Md.) with the following defaults: pairwisealignment, gap opening penalty of 10, gap extension penalty of 0.1, andscore matrix: blosum62mt2.

Amino acid sequence comparisons showed that the Streptococcusequisimilis HasB protein has 70% identity to the HasB protein fromStreptococcus uberis (SEQ ID NO: 105); the Streptococcus equisimilisHasC protein has 91% identity to the HasC protein from Streptococcuspyogenes (SEQ ID NO: 99); and the Streptococcus equisimilis HasD proteinhas 73% identity to the GImU protein (a putative UDP-N-acetylglucosaminepyrophosphorylase) of Streptococcus pyogenes (accession #Q8P286). TheStreptococcus equisimilis hasD gene encodes a polypeptide that shows50.7% identity to the UDP-N-acetyl-glucosamine pyrophosphorylase enzymeencoded by the gcaD gene of Bacillus subtilis.

Plasmid pHA5 (Example 3, FIG. 11) was digested with HpaI and BamHI. Thedigestion was resolved on a 0.8% agarose gel using 0.5×TBE buffer andthe larger vector fragment (approximately 11,000 bp) was gel-purifiedusing the QIAquick DNA Extraction Kit according to the manufacturer'sinstructions. Plasmid pMRT106 was digested with HindIII, the sticky endswere filled in with Klenow fragment, and the DNA was digested withBamHI. The digestion was resolved on a 0.8% agarose gel using 0.5×TBEbuffer and the smaller insert fragment (approximately 1000 bp, the last⅔ of the Streptococcus equisimilis hasD gene) was gel-purified using theQIAquick DNA Extraction kit according to the manufacturer'sinstructions.

The two purified fragments were ligated together with T4 DNA ligase andthe ligation mix was transformed into E. coli SURE competent cells.Transformants were selected on 2×YT agar plates supplemented with 100 μgof ampicillin per ml at 37° C.

Plasmid DNA was purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by BamHI plusNotI digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correctplasmid was identified by the presence of an approximately 1,100 bpBamHI/NotI hasD fragment and was designated pHA8 (FIG. 15). This plasmidwas digested with HindIII and ligated together with T4 DNA ligase andthe ligation mix was transformed into E. coli SURE competent cells.Transformants were selected on 2×YT agar plates supplemented with 100 μgof ampicillin per ml. Plasmid DNA was purified from severaltransformants using a QIAGEN robot according to the manufacturer'sinstructions and analyzed by HindIII digestion on a 0.8% agarose gelusing 0.5×TBE buffer. The correct plasmid was identified by the presenceof a single band of approximately 9,700 bp and was designated pHA9 (FIG.16).

Plasmid pHA9 was digested with SacI and NotI. The digestion was resolvedon a 0.8% agarose gel using 0.5×TBE buffer and the smaller fragment ofapproximately 2,500 bp was gel-purified using the QIAquick DNAExtraction kit according to the manufacturer's instructions. PlasmidpDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digested with SacIand NotI. The digestion was resolved on a 0.8% agarose gel using 0.5×TBEbuffer and the larger vector fragment of approximately 6,800 bp wasgel-purified using the QIAquick DNA Extraction kit according to themanufacturer's instructions. The two purified fragments were ligatedtogether with T4 DNA ligase and the ligation mix was transformed into E.coli SURE competent cells (Stratagene, Inc., La Jolla, Calif.).Transformants were selected on 2×YT agar plates supplemented with 100 μgof ampicillin per ml.

Plasmid DNA was purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by SalI plusHindIII digestion on a 0.8% agarose gel using 0.5×TBE buffer. Thecorrect plasmid was identified by the presence of an approximately 1600bp SalI/HindIII fragment and was designated pHA10 (FIG. 17).

Plasmid pHA10 was digested with HindIII and BamHI. The digestion wasresolved on a 0.8% agarose gel using 0.5×TBE buffer and the largervector fragment (approximately 8100 bp) was gel-purified using theQIAquick DNA Extraction kit according to the manufacturer'sinstructions. Plasmid pMRT106 was digested with HindIII and BamHI. Thedigestion was resolved on a 0.8% agarose gel using 0.5×TBE buffer andthe larger insert fragment of approximately 4,100 bp was gel-purifiedusing the QIAquick DNA Extraction kit according to the manufacturer'sinstructions. The two purified fragments were ligated together with T4DNA ligase and the ligation mix was used to transform Bacillus subtilis16804. Transformants were selected on TBAB agar plates supplemented with5 μg of chloramphenicol per ml at 37° C. Approximately 100 transformantswere patched onto TBAB supplemented with chloramphenicol (5 μg/ml) andTBAB supplemented with neomycin (10 μg/ml) to score chloramphenicolresistant, neomycin sensitive colonies; this phenotype is indicative ofa double crossover into the amyE locus. A few such colonies wereidentified, all of which exhibited a “wet” phenotype indicating thathyaluronic acid was being produced. One colony was chosen and designatedBacillus subtilis 168Δ4::scBAN/se hasA/hasB/hasC/hasD.

Genomic DNA was isolated from Bacillus subtilis 168Δ4::scBAN/sehasA/hasB/hasC/hasD using a QIAGEN tip-20 column according to themanufacturer's instructions, and used to transform competent Bacillussubtilis A164Δ5. Transformants were selected on TBAB plates containing 5μg of chloramphenicol per ml, and grown at 37° C. for 16 hours. TheBacillus subtilis A164Δ5 hasA/hasB/hasC/hasD integrant was identified byits “wet” phenotype and designated Bacillus subtilis TH-1.

Example 8 Construction of Bacillus subtilis RB184

The hasA gene from Streptococcus equisimilis (Example 1) and tuaD gene(a Bacillus subtilis hasB homologue) (Example 1) were cloned to be underthe control of a short “consensus” amyQ (scBAN) promoter (U.S. Pat. No.5,955,310).

Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digestedwith SacI. The digested plasmid was then purified using a QIAquick DNAPurification Kit according to the manufacturer's instructions, andfinally digested with NotI. The largest plasmid fragment ofapproximately 6,800 bp was gel-purified from a 0.8% agarose-0.5×TBE gelusing a QIAquick DNA Gel Extraction Kit according to the manufacturer'sinstructions. The recovered vector DNA was then ligated with the DNAinsert described below.

Plasmid pHA5 (Example 3, FIG. 11) was digested with HpaI. The digestedplasmid was then purified as described above, and finally digested withXbaI. The double-digested plasmid was then blunted by first inactivatingXbaI at 85° C. for 30 minutes. Blunting was performed by adding 0.5 μlof 10 mM each dNTPs, 1 μl of 1 U/μl T4 DNA polymerase (Roche AppliedScience; Indianapolis, Ind.) and incubating at 11° C. for 10 minutes.Finally the polymerase was inactivated by incubating the reaction at 75°C. for 10 minutes. The largest plasmid fragment of approximately 11,000bp was then gel-purified as described above and religated using theRapid DNA Cloning Kit according to the manufacturer's instructions. Theligation mix was transformed into E. coli SURE competent cells.Transformants were selected on 2×YT agar plates supplemented with 100 μgof ampicillin per ml at 37° C. Plasmid DNA was purified from severaltransformants using a QIAGEN robot according to the manufacturer'sinstructions and analyzed by ScaI digestion on a 0.8% agarose gel using0.5×TBE buffer. The correct plasmid was identified by the presence of anapproximately 11 kb fragment and was designated pRB157 (FIG. 18).

pRB157 was digested with SacI. The digested plasmid was then purifiedusing a QIAquick DNA Purification Kit according to the manufacturer'sinstructions, and finally digested with NotI. The smallest plasmidfragment of approximately 2,632 bp was gel-purified using a QIAquick DNAGel Extraction Kit from a 0.8% agarose-0.5×TBE gel according to themanufacturer's instructions. The recovered DNA insert was then ligatedwith the vector DNA described above.

Prior to transformation in Bacillus subtilis, the ligation describedabove was linearized using ScaI to ensure double cross-over integrationin the chromosome rather than single cross-over integration in thechromosome. Bacillus subtilis 168Δ4 competent cells were transformedwith the ligation digested with the restriction enzyme ScaI.

Bacillus subtilis chloramphenicol-resistant transformants were selectedon TBAB plates supplemented with 5 μg of chloramphenicol per ml. Toscreen for integration of the plasmid by double cross-over at the amyElocus, Bacillus subtilis primary transformants were patched on TBABplates supplemented with 6 μg of neomycin per ml and on TBAB platessupplemented with 5 μg of chloramphenicol per ml to isolatechloramphenicol resistant and neomycin sensitive “wet” transformants(due to hyaluronic acid production).

Genomic DNA was isolated from the “wet”, chloramphenicol resistant, andneomycin sensitive Bacillus subtilis 168Δ4 transformants using a QIAGENtip-20 column according to the manufacturer's instructions. PCRamplifications were performed on these transformants using primers 3, 8,and 16 (Example 1) to confirm the presence and integrity of hasA andtuaD in the operon of the Bacillus subtilis transformants. Theamplification reactions (25 μl) were composed of approximately 50 ng ofgenomic DNA of the Bacillus subtilis 168Δ4 transformants, 0.5 μM of eachprimer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR buffer, 3 mMMgCl₂, and 0.625 units of AmpliTaq Gold™ DNA polymerase. The reactionswere incubated in a RoboCycler 40 Temperature Cycler programmed for onecycle at 95° C. for 9 minutes; 30 cycles each at 95° C. for 1 minute,55° C. for 1 minute, and 72° C. for 2 minutes; and a final cycle at 72°C. for 7 minutes.

Primers 3 and 8 were used to confirm the presence of the hasA gene andprimers 3 and 16 to confirm the presence of the tuaD gene. A Bacillussubtilis 168Δ4 hasA/tuaD integrant was designated Bacillus subtilisRB183.

Bacillus subtilis RB183 genomic DNA was also used to transform competentBacillus subtilis A164Δ5. Transformants were selected on TBAB platescontaining 5 μg of chloramphenicol per ml, and grown at 37° C. for 16hours. The Bacillus subtilis A164Δ5 hasA/tuaD integrant was identifiedby its “wet” phenotype and designated Bacillus subtilis RB184.

Example 9 Construction of Bacillus subtilis RB187

Bacillus subtilis RB161 was made competent and transformed with the catdeletion plasmid pRB115 (Widner et al., 2000, Journal of IndustrialMicrobiology & Biotechnology 25: 204-212). Selection for directintegration into the chromosome was performed at the non-permissivetemperature of 45° C. using erythromycin (5 μg/ml) selection. At thistemperature, the pE194 origin of replication is inactive. Cells are ableto maintain erythromycin resistance only by integration of the plasmidinto the cat gene on the bacterial chromosome. These so-called“integrants” were maintained at 45° C. to ensure growth at thistemperature with selection. To allow for loss or “looping out” of theplasmid, which will result in the deletion of most of the cat gene fromthe chromosome, the integrants were grown in Luria-Bertani (LB) mediumwithout selection at the permissive temperature of 34° C. for manygenerations. At this temperature the pE194 origin of replication isactive and promotes excision of the plasmid from the genome (MolecularBiological Methods for Bacillus, edited by C. R. Harwood and S. M.Cutting, 1990, John Wiley and Sons Ltd.).

The cells were then plated on non-selective LB agar plates and colonieswhich contained deletions in the cat gene and loss of the pE194-basedreplicon were identified by the following criteria: (1) chloramphenicolsensitivity indicated the presence of the cat deletion; (2) erythromycinsensitivity indicated the absence of the erythromycin resistance geneencoded by the vector pRB115; and (3) PCR confirmed the presence of thecat deletion in the strain of interest. PCR was performed to confirmdeletion of the cat gene at the amyE locus by using primers 32 and 33:

Primer 32: (SEQ ID NO: 46) 5′-GCGGCCGCGGTACCTGTGTTACACCTGTT-3′Primer 33: (SEQ ID NO: 47) 5′-GTCAAGCTTAATTCTCATGTTTGACAGCTTATCATCGG-3′

Chromosomal DNA from potential deletants was isolated using theREDextract-N-Amp™ Plant PCR kits (Sigma Chemical Company, St. Louis,Mo.) as follows: Single Bacillus colonies were inoculated into 100 μl ofExtraction Solution (Sigma Chemical Company, St. Louis, Mo.), incubatedat 95° C. for 10 minutes, and then diluted with an equal volume ofDilution Solution (Sigma Chemical Company, St. Louis, Mo.). PCR wasperformed using 4 μl of extracted DNA in conjunction with theREDextract-N-Amp PCR Reaction Mix and the desired primers according tothe manufacturer's instructions, with PCR cycling conditions describedin Example 5. PCR reaction products were visualized in a 0.8%agarose-0.5×TBE gel. The verified strain was named Bacillus subtilisRB187.

Example 10 Construction of Bacillus subtilis RB192

Bacillus subtilis RB184 was made unmarked by deleting thechloramphenicol resistance gene (cat gene). This was accomplished usingthe method described previously in Example 9. The resultant strain wasdesignated Bacillus subtilis RB192.

Example 11 Construction of Bacillus subtilis RB194

Bacillus subtilis RB194 was constructed by deleting the cypX region ofthe chromosome of Bacillus subtilis RB187 (Example 9). The cypX regionincludes the cypX gene which encodes a cytochrome P450-like enzyme thatis involved in the synthesis of a red pigment during fermentation. Inorder to delete this region of the chromosome plasmid pMRT086 wasconstructed.

The region of the chromosome which harbors the cypX-yvmC and yvmB-yvmAoperons was PCR amplified from Bacillus subtilis BRG-1 as a singlefragment using primers 34 and 35. Bacillus subtilis BRG1 is essentiallya chemically mutagenized isolate of an amylase-producing strain ofBacillus subtilis which is based on the Bacillus subtilis A164Δ5 geneticbackground that was described in Example 5. The sequence of this regionis identical to the published sequence for the Bacillus subtilis 168type strain.

Primer 34: (SEQ ID NO: 48) 5′-CATGGGAGAGACCTTTGG-3′ Primer 35:(SEQ ID NO: 49) 5′-GTCGGTCTTCCATTTGC-3′

The amplification reactions (50 μl) were composed of 200 ng of Bacillussubtilis BRG-1 chromosomal DNA, 0.4 μM each of primers 34 and 35, 200 μMeach of dATP, dCTP, dGTP, and dTTP, 1× Expand™ High Fidelity buffer(Roche Applied Science; Indianapolis, Ind.) with 1.5 mM MgCl₂, and 2.6units of Expand™ High Fidelity PCR System enzyme mix (Roche AppliedScience; Indianapolis, Ind.). Bacillus subtilis BRG-1 chromosomal DNAwas obtained using a QIAGEN tip-20 column according to themanufacturer's instructions. Amplification reactions were performed in aRoboCycler 40 thermacycler (Stratagene, Inc, La Jolla, Calif.)programmed for 1 cycle at 95° C. for 3 minutes; 10 cycles each at 95° C.for 1 minute, 58° C. for 1 minute, and 68° C. for 4 minutes; 20 cycleseach at 95° C. for 1 minute, 58° C. for 1 minute, 68° C. for 4 minutesplus 20 seconds per cycle, followed by 1 cycle at 72° C. for 7 minutes.Reaction products were analyzed by agarose gel electrophoresis using a0.8% agarose gel using 0.5×TBE buffer.

The resulting fragment comprising the cypX-yvmC and yvmB-yvmA operonswas cloned into pCR2.1 using the TA-TOPO Cloning Kit and transformedinto E. coli OneShot™ cells according to the manufacturer's instructions(Invitrogen, Inc., Carlsbad, Calif.). Transformants were selected on2×YT agar plates supplemented with 100 μg of ampicillin per ml. PlasmidDNA from several transformants was isolated using QIAGEN tip-20 columnsaccording to the manufacturer's instructions and verified by DNAsequencing with M13 (−20) forward, M13 reverse and primers 36 to 51shown below. The resulting plasmid was designated pMRT084 (FIG. 19).

Primer 36: (SEQ ID NO: 50) 5′-CGACCACTGTATCTTGG-3′ Primer 37:(SEQ ID NO: 51) 5′-GAGATGCCAAACAGTGC-3′ Primer 38: (SEQ ID NO: 52)5′-CATGTCCATCGTGACG-3′ Primer 39: (SEQ ID NO: 53)5′-CAGGAGCATTTGATACG-3′ Primer 40: (SEQ ID NO: 54)5′-CCTTCAGATGTGATCC-3′ Primer 41: (SEQ ID NO: 55)5′-GTGTTGACGTCAACTGC-3′ Primer 42: (SEQ ID NO: 56)5′-GTTCAGCCTTTCCTCTCG-3′ Primer 43: (SEQ ID NO: 57)5′-GCTACCTTCTTTCTTAGG-3′ Primer 44: (SEQ ID NO: 58)5′-CGTCAATATGATCTGTGC-3′ Primer 45: (SEQ ID NO: 59)5′-GGAAAGAAGGTCTGTGC-3′ Primer 46: (SEQ ID NO: 60)5′-CAGCTATCAGCTGACAG-3′ Primer 47: (SEQ ID NO: 61)5′-GCTCAGCTATGACATATTCC-3′ Primer 48: (SEQ ID NO: 62)5′-GATCGTCTTGATTACCG-3′ Primer 49: (SEQ ID NO: 63)5′-AGCTTTATCGGTGACG-3′ Primer 50: (SEQ ID NO: 64) 5′-TGAGCACGATTGCAGG-3′Primer 51: (SEQ ID NO: 65) 5′-CATTGCGGAGACATTGC-3′

Plasmid pMRT084 was digested with Bsgl to delete most of the cypX-yvmCand yvmB-yvmA operons, leaving about 500 bases at each end. The digestedBsgl DNA was treated with T4 DNA polymerase. Plasmid pECC1 (Youngman etal., 1984, Plasmid 12: 1-9) was digested with SmaI. A fragment ofapproximately 5,100 bp from pMRT084 and a fragment of approximately1,600 bp fragment from pECC1 were isolated from a 0.8% agarose-0.5×TBEgel using the QIAquick DNA Extraction Kit according to themanufacturer's instructions, ligated together, and transformed into E.coli XL1 Blue cells according to the manufacturer's instructions(Stratagene, Inc., La Jolla, Calif.). Transformants were selected on2×YT agar plates supplemented with 100 μg of ampicillin per ml.Transformants carrying the correct plasmid with most of the cypX-yvmCand yvmB-yvmA operons deleted were identified by PCR amplification usingprimers 52 and 53. PCR amplification was conducted in 50 μl reactionscomposed of 1 ng of plasmid DNA, 0.4 μM of each primer, 200 μM each ofdATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl₂, and 2.5units of AmpliTaq Gold™ DNA polymerase. The reactions were performed ina RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 10minutes; 25 cycles each at 95° C. for 1 minute, 55° C. for 1 minute, and72° C. for 1 minute; and 1 cycle at 72° C. for 7 minutes. The PCRproduct was visualized using a 0.8% agarose-0.5×TBE gel. This constructwas designated pMRT086 (FIG. 20).

Primer 52: (SEQ ID NO: 66) 5′-TAGACAATTGGAAGAGAAAAGAGATA-3′ Primer 53:(SEQ ID NO: 67) 5′-CCGTCGCTATTGTAACCAGT-3′

Plasmid pMRT086 was linearized with ScaI and transformed into Bacillussubtilis RB128 competent cells in the presence of 0.2 μg ofchloramphenicol per ml. Transformants were selected on TBAB platescontaining 5 μg of chloramphenicol per ml after incubation at 37° C. for16 hours. Chromosomal DNA was prepared from several transformants usinga QIAGEN tip-20 column according to the manufacturer's instructions.Chloramphenicol resistant colonies were screened by PCR for deletion ofthe cypX-yvmC and yvmB-yvmA operons via PCR using primers 36 and 52, 36and 53, 37 and 52, and 37 and 53. PCR amplification was conducted in 50μl reactions composed of 50 ng of chromosomal DNA, 0.4 μM of eachprimer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with2.5 mM MgCl₂, and 2.5 units of AmpliTaq Gold™ DNA polymerase. Thereactions were performed in a RoboCycler 40 thermacycler programmed for1 cycle at 95° C. for 10 minutes; 25 cycles each at 95° C. for 1 minute,55° C. for 1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for7 minutes. The PCR products were visualized using a 0.8% agarose-0.5×TBEgel. The resulting Bacillus subtilis RB128 cypX-yvmC and yvmB-yvmAdeleted strain was designated Bacillus subtilis MaTa17.

Competent cells of Bacillus subtilis RB187 (Example 9) were transformedwith genomic DNA from Bacillus subtilis MaTa17. Genomic DNA was obtainedfrom this strain using a QIAGEN tip-20 column according to themanufacturer's instructions. Bacillus subtilis chloramphenicol resistanttransformants were selected on TBAB plates supplemented with 5 μg ofchloramphenicol per ml at 37° C. Primary transformants were streaked forsingle colony isolations on TBAB plates containing 5 μg ofchloramphenicol per ml at 37° C. The resulting cypX-yvmC and yvmB-yvmAdeleted strain was designated Bacillus subtilis RB194.

Example 12 Construction of Bacillus subtilis RB197

Bacillus subtilis RB197 is very similar to Bacillus subtilis RB194, theonly difference being that RB197 contains a smaller deletion in the cypXregion: only a portion of the cypX gene is deleted in this strain togenerate a cypX minus phenotype. In order to accomplish this task aplasmid, pMRT122, was constructed as described below.

Plasmid pCJ791 (FIG. 21) was constructed by digestion of plasmid pSJ2739(WO 96/23073) with EcoRI/HindIII and ligation to a fragment containing adeleted form of the wprA gene (cell wall serine protease) from Bacillussubtilis. The 5′ region of wprA was amplified using primers 54 and 55see below, and the 3′ region was amplified using primers 56 and 57 shownbelow from chromosomal DNA obtained from Bacillus subtilis DN1885(Diderichsen et al., 1990, Journal of Bacteriology 172: 4315-4321). PCRamplification was conducted in 50 μl reactions composed of 1 ng ofBacillus subtilis DN1885 chromosomal DNA, 0.4 μM each of primers 39 and40, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5mM MgCl₂, and 2.5 units of AmpliTaq Gold™ DNA polymerase. The reactionswere performed in a RoboCycler 40 thermacycler programmed for 1 cycle at95° C. for 10 minutes; 25 cycles each at 95° C. for 1 minute, 55° C. for1 minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 7 minutes.

The 5′ and 3′ wprA PCR fragments were linked by digestion with BglIIfollowed by ligation, and PCR amplification was performed on theligation mixture fragments using primers 54 and 57. PCR amplificationwas conducted in 50 μl reactions composed of 1 ng of the ligatedfragment, 0.4 μM of each primer, 200 μM each of dATP, dCTP, dGTP, anddTTP, 1×PCR Buffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaq Gold™DNA polymerase. The reactions were performed in a RoboCycler 40thermacycler programmed for 1 cycle at 95° C. for 10 minutes; 25 cycleseach at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1minute; and 1 cycle at 72° C. for 7 minutes. The PCR product wasvisualized using a 0.8% agarose-0.5×TBE gel. The resulting PCR fragmentwas cloned into pSJ2739 as an EcoRI/HindIII fragment, resulting inplasmid pCJ791 (FIG. 21). Transformants were selected on TBAB-agarplates supplemented with 1 μg of erythromycin and 25 μg of kanamycin perml after incubation at 28° C. for 24-48 hours. Plasmid DNA from severaltransformants was isolated using QIAGEN tip-20 columns according to themanufacturer's instructions and verified by PCR amplification withprimers 54 and 57 using the conditions above.

Primer 54: (SEQ ID NO: 68) 5′-GGAATTCCAAAGCTGCAGCGGCCGGCGCG-3′Primer 55: (SEQ ID NO: 69) 5′-GAAGATCTCGTATACTTGGCTTCTGCAGCTGC-3′Primer 56: (SEQ ID NO: 70) 5′-GAAGATCTGGTCAACAAGCTGGAAAGCACTC-3′Primer 57: (SEQ ID NO: 71) 5′-CCCAAGCTTCGTGACGTACAGCACCGTTCCGGC-3′

The amyL upstream sequence and 5′ coding region from plasmid pDN1981(U.S. Pat. No. 5,698,415) were fused together by SOE using the primerpairs 58/59 and 60/61 shown below. The resulting fragment was clonedinto vector pCR2.1 to generate plasmid pMRT032 as follows. PCRamplifications were conducted in triplicate in 50 μl reactions composedof 1 ng of pDN1981 DNA, 0.4 μM each of appropriate primers, 200 μM eachof dATP, dCTP, dGTP, and dTTP, 1×PCR Buffer II with 2.5 mM MgCl₂, and2.5 units of AmpliTaq Gold™ DNA polymerase. The reactions were performedin a RoboCycler 40 thermacycler programmed for 1 cycle at 95° C. for 9minutes; 3 cycles each at 95° C. for 1 minute, 52° C. for 1 minute, and72° C. for 1 minute; 27 cycles each at 95° C. for 1 minute, 55° C. for 1minute, and 72° C. for 1 minute; and 1 cycle at 72° C. for 5 minutes.The PCR product was visualized in a 0.8% agarose-0.5×TBE gel. Theexpected fragments were approximately 530 and 466 bp, respectively. Thefinal SOE fragment was generated using primer pair 59/60 and cloned intopCR2.1 vector using the TA-TOPO Cloning Kit. Transformants were selectedon 2×YT agar plates supplemented with 100 μg/ml ampicillin afterincubation at 37° C. for 16 hours. Plasmid DNA from severaltransformants was isolated using QIAGEN tip-20 columns according to themanufacturer's instructions and verified by DNA sequencing with M13(−20) forward and M13 reverse primers. The plasmid harboring the amyLupstream sequence/5′ coding sequence fusion fragment was designatedpMRT032 (FIG. 22).

Primer 58: (SEQ ID NO: 72)5′-CCTTAAGGGCCGAATATTTATACGGAGCTCCCTGAAACAACAAAAA CGGC-3′ Primer 59:(SEQ ID NO: 73) 5′-GGTGTTCTCTAGAGCGGCCGCGGTTGCGGTCAGC-3′ Primer 60:(SEQ ID NO: 74) 5′-GTCCTTCTTGGTACCTGGAAGCAGAGC-3′ Primer 61:(SEQ ID NO: 75) 5′-GTATAAATATTCGGCCCTTAAGGCCAGTACCATTTTCCC-3′

Plasmid pNNB194 (pSK⁺/pE194; U.S. Pat. No. 5,958,728) was digested withNsiI and NotI, and plasmid pBEST501 Maya et al. 1989 Nucleic AcidsResearch 17: 4410) was digested with PstI and NotI. The 5,193 bp vectorfragment from pNNB194 and the 1,306 bp fragment bearing the neo genefrom pBEST501 were isolated from a 0.8% agarose-0.5×TBE gel using aQIAquick DNA Purification Kit according to the manufacturer'sinstructions. The isolated fragments were ligated together and used totransform E. coli SURE competent cells according to the manufacturer'sinstructions. Ampicillin-resistant transformants were selected on 2×YTplates supplemented with 100 μg of ampicillin per ml. Plasmid DNA wasisolated from one such transformant using the QIAGEN Plasmid Kit (QIAGENInc., Valencia, Calif.), and the plasmid was verified by digestion withNsiI and NotI. This plasmid was designated pNNB194neo (FIG. 23).

Plasmid pNNB194neo was digested with SacI/NotI and treated with T4 DNApolymerase and dNTPs to generate blunt ends using standard protocols.Plasmid pPL2419 (U.S. Pat. No. 5,958,728) was digested with Ecl136II.The 6,478 bp vector fragment from pNNB194neo and the 562 bp fragmentbearing oriT from pPL2419 were isolated from a 0.8% agarose-0.5×TBE gelusing a QIAquick DNA Purification Kit according to the manufacturer'sinstructions. The gel-purified fragments were ligated together and usedto transform E. coli SURE cells according to the manufacturer'sinstructions. Ampicillin-resistant transformants were selected on 2×YTplates supplemented with 100 μg of ampicillin per ml at 37° C. PlasmidDNA was isolated from one such transformant using the QIAGEN PlasmidKit, and the plasmid was verified by digestion with NSiI, SacI, andBscI. This plasmid was designated pNNB194neo-oriT (FIG. 24).

Plasmid pNNB194neo-oriT was digested with BamHI and treated with T4 DNApolymerase and dNTPs to generate blunt ends using standard protocols.The digested plasmid was gel-purified from a 0.8% agarose-0.5×TBE gelusing a QIAquick DNA Purification Kit according to the manufacturer'sinstructions. The purified plasmid was treated with T4 DNA ligase andused to transform E. coli SURE cells according to the manufacturer'sinstructions. Ampicillin-resistant transformants were selected on 2×YTplates supplemented with 100 μg of ampicillin per ml at 37° C. PlasmidDNA was isolated from one such transformant using the QIAGEN PlasmidKit, and disruption of the BamHI site was confirmed by digestion withBamHI and ScaI. The resulting plasmid was designated pShV3 (FIG. 25).

Plasmid pShV2.1-amyEA (U.S. Pat. No. 5,958,728) was digested with SfiIand NotI, and the 8696 bp vector fragment was gel-purified from a 0.8%agarose-0.5×TBE gel using a QIAquick DNA Purification Kit according tothe manufacturer's instructions. In order to insert a BamHI site betweenthe SfiI and NotI sites of pShV2.1-amyEA, a synthetic linker wasconstructed as follows: primers 62 and 63 were annealed by mixing 50 μMof each, boiling the mixture, and allowing the mixture to cool slowly.

Primer 62: (SEQ ID NO: 76) 5′-GGGCCGGATCCGC-3′ Primer 63:(SEQ ID NO: 77) 3′-ATTCCCGGCCTAGGCGCCGG-5′

The purified pShV2.1-amyEA vector and annealed oligonucleotides wereligated together and used to transform E. coli SURE competent cellsaccording to the manufacturer's instructions. Chloramphenicol-resistanttransformants were selected on LB plates supplemented with 30 μg ofchloramphenicol per ml at 37° C. Plasmid DNA was isolated from one suchtransformant using the QIAGEN Plasmid Kit, and insertion of the BamHIsite was confirmed by digestion with BamHI. This plasmid was designatedpShV2.1-amyEΔB (FIG. 26).

Plasmids pShV3 and pShV2.1-amyEΔB were digested with SalI/HindIII. A7033 bp vector fragment from pShV3 and a 1031 bp fragment bearing amyEΔ,from pShV2.1-amyEΔ were gel-purified from a 0.8% agarose-0.5×TBE gelusing a QIAquick DNA Purification Kit according to the manufacturer'sinstructions. The gel-purified fragments were ligated together and usedto transform E. coli SURE cells according to the manufacturer'sinstructions. Ampicillin-resistant transformants were selected on 2×YTplates supplemented with 100 μg of ampicillin per ml. Plasmid DNA wasisolated from one such transformant using the QIAGEN Plasmid Kit, andthe plasmid was verified by digestion with SalI and HindIII. Thisplasmid was designated pShV3A (FIG. 27).

Plasmid pMRT032 was digested with KpnI/XbaI, filled with Klenow fragmentDNA polymerase in the presence of dNTPs, and a fragment of approximately1000 bp was isolated from a 0.8% agarose-0.5×TBE gel using a QIAquickDNA Purification Kit according to the manufacturer's instructions. Thisfragment was cloned into plasmid pShV3a digested with EcoRV, andtransformed into E. coli XL1 Blue cells according to the manufacturer'sinstructions. Transformants were selected on 2×YT agar platessupplemented with 100 μg of ampicillin per ml after incubation at 37° C.for 16 hours. Plasmid DNA from several transformants was isolated usingQIAGEN tip-20 columns according to the manufacturer's instructions andverified on a 0.8% agarose-0.5×TBE gel by restriction analysis withSacI/SphI. The resulting plasmid was designated pMRT036 (FIG. 28).

Plasmid pMRT036 was digested with SalI/HindIII, filled with Klenowfragment DNA polymerase in the presence of dNTPs, ligated andtransformed into E. coli XL1 Blue cells according to the manufacturer'sinstructions. Transformants were selected on 2×YT-agar platessupplemented with 100 μg/ml ampicillin after incubation at 37° C. for 16hours. Plasmid DNA from several transformants was isolated using QIAGENtip-20 columns according to the manufacturer's instructions and verifiedon a 0.8% agarose-0.5×TBE gel by restriction analysis with SacI/XbaI,PstI and NdeI. The resulting plasmid was designated pMRT037 (FIG. 29).

The scBAN/cryIIIA stabilizer fragment from plasmidpDG268Δneo-cryIIIAstab/Sav (U.S. Pat. No. 5,955,310) was isolated from a2% agarose-0.5×TBE gel as a SfiI/SacI fragment using a QIAquick DNAPurification Kit according to the manufacturer's instructions, ligatedto plasmid pMRT037 digested with SfiI/SacI, and transformed into E. coliXL1 Blue cells. Transformants were selected on 2×YT agar platessupplemented with 100 μg of ampicillin per ml after incubation at 37° C.for 16 hours. Plasmid DNA from several transformants was isolated usingQIAGEN tip-20 columns according to the manufacturer's instructions andverified on a 0.8% agarose-0.5×TBE gel by restriction analysis withPstI. The resulting plasmid was designated pMRT041 (FIG. 30).

Plasmids pMRT041 and pCJ791 were digested with EcoRI/HindIII. A fragmentof approximately 1300 bp from pMRT041 and a fragment of approximately4500 bp from pCJ791 were isolated from a 0.8% agarose-0.5×TBE gel usinga QIAquick DNA Purification Kit according to the manufacturer'sinstructions, ligated, and transformed into Bacillus subtilis 168Δ4competent cells. Transformants were selected on TBAB-agar platessupplemented with 1 μg of erythromycin and 25 μg of lincomycin per mlafter incubation at 30° C. for 24-48 hours. Plasmid DNA from severaltransformants was isolated using QIAGEN tip-20 columns according to themanufacturer's instructions and verified on a 0.8% agarose-0.5×TBE gelby restriction analysis with SacI and EcoRI/HindIII. The resultingplasmid was designated pMRT064.1 (FIG. 31).

The SacI site at position 2666 in plasmid pMRT064.1 was deleted by SOEusing primer pairs 64 and 65, and primer pairs 66 and 67 shown below.PCR amplification was conducted in 50 μl reactions composed of 1 ng ofpMRT064.1 DNA, 0.4 μM of each primer, 200 μM each of dATP, dCTP, dGTP,and dTTP, 1×PCR Buffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaqGold™ DNA polymerase. The reactions were performed in a RoboCycler 40thermacycler programmed for 1 cycle at 95° C. for 10 minutes; 25 cycleseach at 95° C. for 1 minute, 52° C. for 1 minute, and 72° C. for 1minute; and 1 cycle at 72° C. for 7 minutes. The PCR product wasvisualized in a 0.8% agarose-0.5×TBE gel. The expected fragments wereapproximately 400 and 800 bp, respectively. The final fragment forcloning back into pMRT064.1 was amplified using primers 64 and 67. Thisfragment was cloned into pCR2.1 vector using the TA-TOPO Cloning Kit.Transformants were selected on 2×YT agar plates supplemented with 100μg/ml ampicillin after incubation at 37° C. for 16 hours. Transformantscarrying the correct plasmid were verified by DNA sequencing using M13forward and reverse primers, and primers 65, 67, and 68. This plasmidwas designated pMRT068 (FIG. 32), and was further transformed into E.coli DM1 cells (Stratagene, Inc., La Jolla, Calif.) according to themanufacturer's instructions. Transformants were selected on 2×YT agarplates supplemented with 100 μg of ampicillin per ml.

Primer 64: (SEQ ID NO: 78) 5′-GGAAATTATCGTGATCAAC-3′ Primer 65:(SEQ ID NO: 79) 5′-GCACGAGCACTGATAAATATG-3′ Primer 66: (SEQ ID NO: 80)5′-CATATTTATCAGTGCTCGTGC -3′ Primer 67: (SEQ ID NO: 81)5′-TCGTAGACCTCATATGC-3′ Primer 68: (SEQ ID NO: 82)5′-GTCGTTAAACCGTGTGC-3′

The SacI sites at positions 5463 and 6025 in plasmid pMRT064.1 weredeleted using PCR amplification with primers 69 and 70, and using thePCR conditions described above. The resulting fragment was cloned intopCR2.1 vector using the TA-TOPO Cloning Kit (Invitrogen, Inc., Carlsbad,Calif.). Transformants were selected on 2×YT-agar plates supplementedwith 100 μg of ampicillin per ml after incubation at 37° C. for 16hours. Transformants carrying the correct plasmid were verified by DNAsequencing using M13 forward and reverse primers. This construct wasdesignated pMRT069 (FIG. 33).

Primer 69: (SEQ ID NO: 83) 5′-CTAGAGGATCCCCGGGTACCGTGCTCTGCCTTTTAGTCC-3′Primer 70: (SEQ ID NO: 84) 5′-GTACATCGAATTCGTGCTCATTATTAATCTGTTCAGC-3′

Plasmids pMRT068 and pMRT064.1 were digested with BclI/AccI. A fragmentof approximately 1300 bp from pMRT068 and a fragment of approximately3800 bp from pMRT064.1 were isolated from a 0.8% agarose-0.5×TBE gelusing a QIAquick DNA Purification Kit according to the manufacturer'sinstructions, ligated, and transformed into Bacillus subtilis 168Δ4competent cells. Transformants were selected on TBAB-agar platessupplemented with 1 μg of erythromycin and 25 μg of lincomycin per mlafter incubation at 30° C. for 24-48 hours. Transformants carrying thecorrect plasmid were identified on a 0.8% agarose-0.5×TBE gel byrestriction analysis with SacI and EcoRI/AvaI. The resulting constructwas designated pMRT071 (FIG. 34).

Plasmids pMRT071 and pMRT069 were digested with AvaI/EcoRI. The 578 bpfragment from pMRT069 and the 4510 bp fragment from pMRT071 wereisolated from a 0.8% agarose-0.5×TBE gel using a QIAquick DNAPurification Kit according to the manufacturer's instructions, ligated,and transformed into Bacillus subtilis 168Δ4 competent cells.Transformants were selected on TBAB-agar plates supplemented with 1 μgof erythromycin and 25 μg of lincomycin per ml after incubation at 30°C. for 24-48 hours. Transformants carrying the correct plasmid wereidentified on a 0.8% agarose-0.5×TBE gel by restriction analysis withSacI. The resulting construct was designated pMRT074 (FIG. 35).

Plasmid pMRT084 described in Example 11 was digested with SacII/NdeI,treated with T4 DNA polymerase, ligated, and transformed into E. coliXL1 Blue cells according to the manufacturer's instructions.Transformants were selected on 2×YT agar plates supplemented with 100 μgof ampicillin per ml after incubation at 37° C. for 16 hours.Transformants carrying the correct plasmid were identified on a 0.8%agarose-0.5×TBE gel by restriction analysis with DraI. The resultingplasmid was named pMRT120 (FIG. 36).

Plasmid pMRT074 was digested with HindIII, treated with Klenow fragmentDNA polymerase, and digested with EcoRI. Plasmid pMRT120 was digestedwith EcoRI/Ecl136II. A fragment of approximately 600 bp from pMRT120 anda fragment of approximately 4300 bp from pMRT074 were isolated from a0.8% agarose-0.5×TBE gel using a QIAquick DNA Purification Kit accordingto the manufacturer's instructions, ligated, and transformed intoBacillus subtilis 168Δ4 competent cells. Transformants were selected onTBAB-agar plates supplemented with 1 μg of erythromycin and 25 μg oflincomycin per ml after incubation at 30° C. for 24-48 hours.Transformants carrying the correct plasmid were identified on a 0.8%agarose-0.5×TBE gel by restriction analysis with SspI. The resultingconstruct was designated pMRT122 (FIG. 37).

Plasmid pMRT122 was transformed into Bacillus subtilis A164Δ5 competentcells. Transformants were selected on TBAB-agar plates supplemented with1 μg of erythromycin and 25 μg of lincomycin per ml after incubation at30° C. for 24-48 hours. The plasmid was introduced into the chromosomeof Bacillus subtilis A164Δ5 via homologous recombination into the cypXlocus by incubating a freshly streaked plate of Bacillus subtilis A164Δ5(pMRT086) cells at 45° C. for 16 hours and selecting for healthy growingcolonies. Genomic DNA was isolated from this strain using a QIAGENtip-20 column according to the manufacturer's instructions and used totransform Bacillus subtilis RB187 (Example 9). Transformants wereselected on TBAB plates supplemented with 1 μg of erythromycin and 25 μgof lincomycin per ml after incubation at 45° C. for 16 hours. At thistemperature, the pE194 replicon is unable to replicate. Cells are ableto maintain erythromycin resistance only by maintaining the plasmid inthe bacterial chromosome.

The plasmid was removed from the chromosome via homologous recombinationresulting in the deletion of a portion of the cypX gene on thechromosome by growing the transformants in Luria-Bertani (LB) mediumwithout selection at the permissive temperature of 34° C. for manygenerations. At this temperature the pE194 origin of replication isactive and actually promotes the excision of the plasmid from thechromosome (Molecular Biological Methods for Bacillus, edited by C. R.Harwood and S. M. Cutting, 1990, John Wiley and Sons Ltd.).

After several generations of outgrowth the cells were plated onnon-selective LB agar plates and colonies which had lost the plasmid andwere now cypX-deleted and producing hyaluronic acid were identified asfollows: (1) cell patches were “wet” when plated on minimal platesindicating production of hyaluronic acid, (2) erythromycin sensitivityindicated loss of the pE194-based plasmid, and (3) PCR confirmed thepresence of the 800 bp cypX deletion in the strain of interest by usingprimers 34 and 45.

Chromosomal DNA from potential cypX deletants was isolated using theREDextract-N-Amp™ Plant PCR kits as follows: Single Bacillus colonieswere inoculated into 100 μl of Extraction Solution, incubated at 95° C.for 10 minutes, and then diluted with an equal volume of DilutionSolution. PCR was performed using 4 μl of extracted DNA in conjunctionwith the REDextract-N-Amp™ PCR Reaction Mix and the desired primersaccording to the manufacturer's instructions, using PCR cyclingconditions as described in Example 5. PCR reaction products werevisualized using a 0.8% agarose-0.5×TBE gel. The verified strain wasdesignated Bacillus subtilis RB197.

Example 13 Construction of Bacillus subtilis RB200

The cypX gene of Bacillus subtilis RB192 was deleted using the samemethods described in Example 9 for Bacillus subtilis RB187. Theresultant strain was designated Bacillus subtilis RB200.

Example 14 Construction of Bacillus subtilis RB202

Bacillus subtilis A164Δ5ΔcypX was constructed as follows: PlasmidpMRT122 (Example 12) was transformed into Bacillus subtilis A164Δ5competent cells. Transformants were selected on TBAB-agar platessupplemented with 1 μg of erythromycin and 25 μg of lincomycin per mlafter incubation at 30° C. for 24-48 hours. The plasmid was introducedinto the chromosome of Bacillus subtilis A164Δ5 via homologousrecombination into the cypX locus by incubating a freshly streaked plateof Bacillus subtilis A164Δ5 (pMRT086) cells at 450 for 16 hours andselecting for healthy growing colonies. The plasmid was removed from thechromosome via homologous recombination resulting in the deletion of aportion of the cypX gene on the chromosome by growing the transformantsin Luria-Bertani (LB) medium without selection at the permissivetemperature of 34° C. for many generations. At this temperature thepE194 origin of replication is active and actually promotes the excisionof the plasmid from the chromosome (Molecular Biological Methods forBacillus, edited by C. R. Harwood and S. M. Cutting, 1990, John Wileyand Sons Ltd.). After several generations of outgrowth the cells wereplated on non-selective LB agar plates and colonies which had lost theplasmid and were now cypX-deleted were identified as follows: (1)erythromycin sensitivity indicated loss of the pE194-based plasmid, and(2) PCR confirmed the presence of the 800 bp cypX deletion in the strainof interest by using primers 34 and 45 as described above. The verifiedstrain was designated Bacillus subtilis A164□5□cypX.

Bacillus subtilis A164Δ5ΔcypX was made competent and transformed withBacillus subtilis TH1 genomic DNA (Example 7) isolated using a QIAGENtip-20 column according to the manufacturer's instructions.Transformants were selected on TBAB plates containing 5 μg ofchloramphenicol per ml at 37° C. The Bacillus subtilis A164Δ5ΔcypXhasA/hasB/hasC/hasD integrant was identified by its “wet” phenotype anddesignated Bacillus subtilis RB201. The cat gene was deleted fromBacillus subtilis RB201 using the same method described in Example 9.The resultant strain was designated Bacillus subtilis RB202.

Example 15 Construction of Bacillus subtilis MF002 (tuaD/gtaB)

Plasmid pHA3 (Example 2, FIG. 9) was digested with Asp718. The digestedplasmid was then blunted by first inactivating the restriction enzyme at85° C. for 30 minutes. Blunting was performed by adding 0.5 μl of 10 mMeach dNTPs, 1 μl of 1 U/μl T4 polymerase and incubating at 11° C. for 10minutes. Finally the polymerase was inactivated by incubating thereaction at 75° C. for 10 minutes. The digested plasmid was thenpurified using a QIAquick DNA Purification Kit according to themanufacturer's instructions and finally digested with NotI. The smallestplasmid fragment of approximately 2522 bp was then gel-purified using aQIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gelaccording to the manufacturer's instructions. The recovered DNA insert(tuaD/gtaB) was then ligated with the vector DNA described below.

Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) was digestedwith Ecl136II. The digested plasmid was then purified using a QIAquickDNA Purification Kit according to the manufacturer's instructions, andfinally digested with NotI. The largest plasmid fragment ofapproximately 6800 bp was gel-purified from a 0.8% agarose-0.5×TBE gelusing a QIAquick DNA Gel Extraction Kit according to the manufacturer'sinstructions.

The recovered vector and DNA insert were ligated using the Rapid DNACloning Kit according to the manufacturer's instructions. Prior totransformation in Bacillus subtilis, the ligation described above waslinearized using ScaI to ensure double cross-over integration in thechromosome rather than single cross-over integration in the chromosome.Bacillus subtilis 168Δ4 competent cells were transformed with theligation digested with the restriction enzyme ScaI.

Bacillus subtilis chloramphenicol-resistant transformants were selectedon TBAB plates supplemented with 5 μg of chloramphenicol per ml. Toscreen for integration of the plasmid by double cross-over at the amyElocus, Bacillus subtilis primary transformants were patched on TBABplates supplemented with 6 μg of neomycin per ml and on TBAB platessupplemented with 5 μg of chloramphenicol per ml to isolatechloramphenicol resistant and neomycin sensitive transformants wereisolated.

Chromosomal DNA from chloramphenicol resistant and neomycin sensitiveBacillus subtilis 168Δ4 transformants was isolated using theREDextract-N-Amp™ Plant PCR kits (Sigma Chemical Company, St. Louis,Mo.) as follows: Single Bacillus colonies were inoculated into 100 μl ofExtraction Solution, incubated at 95° C. for 10 minutes, and thendiluted with an equal volume of Dilution Solution. PCR was performedusing 4 μl of extracted DNA in conjunction with the REDextract-N-Amp PCRReaction Mix and the desired primers according to the manufacturer'sinstructions, with PCR cycling conditions described in Example 5.

PCR amplifications were performed on these transformants using thesynthetic oligonucleotides described below to confirm theabsence/presence and integrity of the genes hasA, gtaB, and tuaD of theoperon of the Bacillus subtilis transformants. Primers 3 and 8 were usedto confirm the absence of the hasA gene, primer 71 and primer 15 toconfirm the presence of the tuaD gene, and primers 20 and 71 to confirmthe presence of the gtaB gene. PCR reaction products were visualized ina 0.8% agarose-0.5×TBE gel. The verified strain, a Bacillus subtilis168Δ4 hasA/tuaD/gtaB integrant, was designated Bacillus subtilis RB176.Primer 71: 5′-AACTATTGCCGATGATAAGC-3′ (binds upstream of tuaD) (SEQ IDNO: 85)

Genomic DNA was isolated from the chloramphenicol resistant, andneomycin sensitive Bacillus subtilis RB176 transformants using a QIAGENtip-20 column according to the manufacturer's instructions. The Bacillussubtilis RB176 genomic DNA was used to transform competent Bacillussubtilis A164Δ5. Transformants were selected on TBAB plates containing 5μg of chloramphenicol per ml, and grown at 37° C. A Bacillus subtilisA164Δ5 tuaD/gtaB integrant was designated Bacillus subtilis RB177.

The cat gene was deleted in strain Bacillus subtilis RB177 using themethod described in Example 9. The resultant strain was designatedBacillus subtilis MF002.

Example 16 Construction of the pel Integration Plasmid pRB162

Plasmid pDG268MCSΔneo/scBAN/Sav (U.S. Pat. No. 5,955,310) wasdouble-digested with SacI and AatII. The largest plasmid fragment ofapproximately 6193 bp was gel-purified using a QIAquick DNA GelExtraction Kit from a 0.8% agarose-0.5×TBE gel according to themanufacturer's instructions. The recovered vector DNA was then ligatedwith the DNA insert described below.

The 5′ and 3′ fragments of a Bacillus subtilis pectate lyase gene (pel,accession number BG10840, SEQ ID NOs. 86 [DNA sequence] and 87 [deducedamino acid sequence]) was PCR amplified from Bacillus subtilis 168 (BGSC1A1, Bacillus Genetic Stock Center, Columbus, Ohio) using primers 72(introduces 5′ SpeI restriction site) and 73 (introduces 3′ SalIrestriction site) for the 5′ pel fragment and primers 74 (introduces 5′SacI/BamHI restriction sites) and 75 (introduces 3′ NotI/AatIIrestriction sites) for the 3′ pel fragment:

Primer 72: (SEQ ID NO: 88) 5′-ACTAGTAATGATGGCTGGGGCGCGTA-3′ Primer 73:(SEQ ID NO: 89) 5′-GTCGACATGTTGTCGTATTGTGAGTT-3′ Primer 74:(SEQ ID NO: 90) 5′-GAGCTCTACAACGCTTATGGATCCGCGGCCGCGGCGGCACACACATCTGGAT-3′ Primer 75: (SEQ ID NO: 91) 5′-GACGTCAGCCCGTTTGCAGCCGATGC-3′

PCR amplifications were carried out in triplicate in 30 μl reactionscomposed of 50 ng of Bacillus subtilis 168 chromosomal DNA, 0.4 μM eachof primer pair 72/73 for the 5′ pel fragment or primer pair 74/75 forthe 3′ pel fragment, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×PCRBuffer II with 2.5 mM MgCl₂, and 2.5 units of AmpliTaq Gold™ DNApolymerase. The reactions were performed in a RoboCycler 40 thermacyclerprogrammed for 1 cycle at 95° C. for 9 minutes; 3 cycles each at 95° C.for 1 minute, 52° C. for 1 minute, and 72° C. for 1 minute; 27 cycleseach at 95° C. for 1 minute, 55° C. for 1 minute, and 72° C. for 1minute; and 1 cycle at 72° C. for 5 minutes. The PCR products werevisualized using a 0.8% agarose-0.5×TBE gel. The expected fragments wereapproximately 530 bp for the 5′ pel fragment and 530 bp for the 3′ pelfragment.

The 530 bp 5′ pel and 530 bp 3′ pel PCR fragments were cloned intopCR2.1 using the TA-TOPO Cloning Kit and transformed into E. coliOneShot™ competent cells according to the manufacturers' instructions.Transformants were selected on 2×YT agar plates supplemented with 100 μgof ampicillin per ml incubated at 37° C. Plasmid DNA from thesetransformants was purified using a QIAGEN robot according to themanufacturer's instructions and the DNA sequence of the insertsconfirmed by DNA sequencing using the primers described above (primers72 and 73 for 5′ pel and primers 74 and 75 for 3′ pel). The plasmidsharboring the 530 bp and the 530 bp PCR fragments were designatedpCR2.1-pel 5′ and pCR2.1-pel3′, respectively (FIGS. 38 and 39,respectively).

Plasmid pCR2.1-pel3′ was double-digested with SacI and AatII. Thesmallest plasmid fragment of approximately 530 bp was gel-purified usinga QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gelaccording to the manufacturer's instructions.

The recovered vector (pDG268MCSΔneo/scBAN) and DNA insert (3′ pel) wereligated using the Rapid DNA Cloning Kit according to the manufacturer'sinstructions. The ligation mix was transformed into E. coli SUREcompetent cells (Stratagene, Inc., La Jolla, Calif.). Transformants wereselected on 2×YT agar plates supplemented with 100 μg of ampicillin perml at 37° C.

Plasmid DNA was purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by SacI andAatII digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correctplasmid was identified by the presence of an approximately 530 bpSacI/AatII 3′ pel fragment and was designated pRB161 (FIG. 40).

Plasmid pRB161 was double-digested with SpeI and SalI. The largestplasmid fragment of approximately 5346 bp was gel-purified using aQIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gelaccording to the manufacturer's instructions. The recovered vector DNAwas then ligated with the DNA insert described below.

Plasmid pCR2.1-pel5′ was double-digested with SpeI and SalI. Thesmallest plasmid fragment of approximately 530 bp was gel-purified usinga QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gelaccording to the manufacturer's instructions.

The recovered vector (pDG268MCSΔneo/scBAN/pel 3′) and insert (pel 5′)DNA were ligated using the Rapid DNA Cloning Kit according to themanufacturer's instructions. The ligation mix was transformed into E.coli SURE competent cells (Stratagene, Inc., La Jolla, Calif.).Transformants were selected on 2×YT agar plates supplemented with 100 μgof ampicillin per ml.

Plasmid DNA was purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by SpeI andSalI digestion on a 0.8% agarose gel using 0.5×TBE buffer. The correctplasmid was identified by the presence of an approximately 530 bpSpeI/SalI pel 5′ fragment and was designated pRB162 (FIG. 41).

Example 17 Construction of pRB156

Plasmid pHA7 (Example 4, FIG. 13) was digested with HpaI. The digestedplasmid was then purified using a QIAquick DNA Purification Kitaccording to the manufacturer's instructions and finally digested withAsp718. The double-digested plasmid was then blunted by firstinactivating the restriction enzyme at 85° C. for 30 minutes. Bluntingwas performed by adding 0.5 μl of 10 mM each dNTPs and 1 μl of 1 U/μl ofT4 polymerase and incubating at 11° C. for 10 minutes. The polymerasewas then inactivated by incubating the reaction at 75° C. for 10minutes. The largest plasmid fragment of approximately 8600 bp was thengel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8%agarose-0.5×TBE gel according to the manufacturer's instructions. Therecovered DNA insert (pDG268Δneo-cryIIIAstab/sehasA) was then re-ligatedusing the Rapid DNA Cloning Kit according to the manufacturer'sinstructions.

The ligation mix was transformed into E. coli SURE competent cells(Stratagene, Inc., La Jolla, Calif.). Transformants were selected on2×YT agar plates supplemented with 100 μg of ampicillin per ml at 37° C.Plasmid DNA was purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by ScaIdigestion on a 0.8% agarose gel using 0.5×TBE buffer. The correctplasmid was identified by the presence of an approximately 8,755 bpfragment and was designated pRB156 (FIG. 42).

Example 18 Construction of Bacillus subtilis MF009

The hasA gene under control of the scBAN promoter was introduced intothe pectate lyase gene (pel) locus of Bacillus subtilis MF002 togenerate Bacillus subtilis MF009.

Plasmid pRB156 was digested with SacI. The digested plasmid was thenpurified using a QIAquick DNA Purification Kit according to themanufacturer's instructions, and finally digested with NotI. Thesmallest plasmid fragment of approximately 1,377 bp was gel-purifiedusing a QIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gelaccording to the manufacturer's instructions. The recovered DNA insertwas then ligated with the vector DNA described below.

Plasmid pRB162 (Example 16, FIG. 41) was digested with NotI. Thedigested plasmid was then purified using a QIAquick DNA Purification Kitaccording to the manufacturer's instructions, and finally digested withSacI. The largest plasmid fragment of approximately 5850 bp wasgel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8%agarose-0.5×TBE gel according to the manufacturer's instructions. Therecovered vector DNA was then ligated with the DNA insert describedabove.

The ligation mixture was transformed directly in Bacillus subtilis 168Δ4competent cells. Bacillus subtilis chloramphenicol-resistanttransformants were selected on TBAB plates supplemented with 5 μg ofchloramphenicol per ml at 37° C. To screen for integration of theplasmid by double cross-over at the pel locus, Bacillus subtilis primarytransformants were patched on TBAB plates supplemented with 6 μg ofneomycin per ml and on TBAB plates supplemented with 5 μg ofchloramphenicol per ml. Integration of the plasmid by double cross-overat the pel locus does not incorporate the neomycin resistance gene andtherefore renders the strain neomycin sensitive. Using this platescreen, chloramphenicol resistant and neomycin sensitive transformantswere isolated.

Genomic DNA was isolated from the chloramphenicol resistant and neomycinsensitive Bacillus subtilis 168Δ4 transformants using a QIAGEN tip-20column according to the manufacturer's instructions. This genomic DNAwas used to transform competent Bacillus subtilis MF002 (Example 15).Transformants were selected on TBAB plates containing 5 μg ofchloramphenicol per ml and grown at 37° C. The Bacillus subtilis A164Δ5hasA and tuaD/gtaB integrant was identified by its “wet” phenotype anddesignated Bacillus subtilis MF009.

Example 19 Construction of Bacillus subtilis MF010

Plasmid pDG268MCSΔneo/BAN/Sav (U.S. Pat. No. 5,955,310) was digestedwith NotI. The digested plasmid was then purified using a QIAquick DNAPurification Kit according to the manufacturer's instructions, andfinally digested with SfiI. The smallest plasmid fragment ofapproximately 185 bp was gel-purified using a QIAquick DNA GelExtraction Kit from a 0.8% agarose-0.5×TBE gel according to themanufacturer's instructions. The recovered DNA insert was then ligatedwith the vector DNA described below.

Plasmid pRB162 (Example 16, FIG. 41) was digested with NotI. Thedigested plasmid was then purified using a QIAquick DNA Purification Kitaccording to the manufacturer's instructions, and finally digested withSfiI. The largest plasmid fragment of approximately 5747 bp wasgel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8%agarose-0.5×TBE gel according to the manufacturer's instructions. Therecovered vector DNA was then ligated with the DNA insert describedabove.

The recovered vector and DNA insert were ligated using the Rapid DNACloning Kit according to the manufacturer's instructions. The ligationmix was transformed into E. coli XLI Blue competent cells. Transformantswere selected on 2×YT agar plates supplemented with 100 μg of ampicillinper ml.

Plasmid DNA was purified from several transformants using a QIAGEN robotaccording to the manufacturer's instructions and analyzed by BamHIdigestion on a 0.8% agarose gel using 0.5×TBE buffer. The correctplasmid was identified by the linearization of the plasmid whichprovides an approximately 7,156 bp fragment and was designated pRB164(FIG. 43).

Plasmid pRB156 (Example 17, FIG. 42) was digested with SacI. Thedigested plasmid was then purified using a QIAquick DNA Purification Kitaccording to the manufacturer's instructions, and finally digested withNotI. The smallest plasmid fragment of approximately 1377 bp wasgel-purified using a QIAquick DNA Gel Extraction Kit from a 0.8%agarose-0.5×TBE gel according to the manufacturer's instructions. Therecovered DNA insert was then ligated with the vector DNA describedbelow.

Plasmid pRB164 was digested with NotI. The digested plasmid was thenpurified using a QIAquick DNA Purification Kit according to themanufacturer's instructions, and finally digested with SacI. The largestplasmid fragment of approximately 5922 bp was gel-purified using aQIAquick DNA Gel Extraction Kit from a 0.8% agarose-0.5×TBE gelaccording to the manufacturer's instructions. The recovered vector DNAwas then ligated with the DNA insert described above.

This ligation mix was transformed directly in Bacillus subtilis 168Δ4competent cells. Bacillus subtilis chloramphenicol-resistanttransformants were selected on TBAB plates supplemented with 5 μg ofchloramphenicol per ml at 37° C. To screen for integration of theplasmid by double cross-over at the amyE locus, Bacillus subtilisprimary transformants were patched on TBAB plates supplemented with 6 μgof neomycin per ml and on TBAB plates supplemented with 5 μg ofchloramphenicol per ml. Integration of the plasmid by double cross-overat the amyE locus does not incorporate the neomycin resistance gene andtherefore renders the strain neomycin sensitive. Using this platescreen, chloramphenicol resistant and neomycin sensitive transformantswere isolated.

Genomic DNA was isolated from the chloramphenicol resistant and neomycinsensitive Bacillus subtilis 168Δ4 transformants using a QIAGEN tip-20column according to the manufacturer's instructions. This genomic DNAwas used to transform competent Bacillus subtilis MF002 (Example 15).Transformants were selected on minimal plates containing 5 μg ofchloramphenicol per ml and grown at 37° C. for 16 hours. A Bacillussubtilis A164Δ5 BAN/hasA and scBAN/tuaD/gtaB integrant was identified byits “wet” phenotype and designated Bacillus subtilis MF010.

Example 20 Fermentations

The ability of the Bacillus subtilis strains listed in Table 1 toproduce hyaluronic acid was evaluated under various growth conditions.

TABLE 1 B. subtilis Strain promoter/gene complement catΔ cypXΔ RB161scBAN/hasA/tuaD/gtaB no no RB163 scBAN/hasA/tuaD/gcaD no no TH-1scBANhasA/hasB/hasC/hasD no no RB184 scBAN/hasA/tuaD no no RB187scBAN/hasA/tuaD/gtaB yes no RB192 scBAN/hasA/tuaD yes no RB194scBAN/hasA/tuaD/gtaB yes yes RB197 scBAN/hasA/tuaD/gtaB yes yes RB200scBAN/hasA/tuaD yes yes RB202 scBAN/hasA/hasB/hasC/hasD yes yes MF009scBAN/tuaD/gtaB no no scBAN/hasA MF010 scBAN/tuaD/gtaB no no BAN/hasA

The Bacillus subtilis strains were fermented in standard smallfermenters in a medium composed per liter of 6.5 g of KH₂PO₄, 4.5 g ofNa₂HPO₄, 3.0 g of (NH₄)₂SO₄, 2.0 g of Na₃-citrate-2H₂O, 3.0 g ofMgSO₄.7H₂O, 6.0 ml of Mikrosoy-2, 0.15 mg of biotin (1 ml of 0.15 mg/mlethanol), 15.0 g of sucrose, 1.0 ml of SB 2066, 2.0 ml of P2000, 0.5 gof CaCl₂.2H₂O. The medium was pH 6.3 to 6.4 (unadjusted) prior toautoclaving. The CaCl₂.2H₂O was added after autoclaving.

The seed medium used was B-3, i.e., Agar-3 without agar, or “S/S-1”medium. The Agar-3 medium was composed per liter of 4.0 g of nutrientbroth, 7.5 g of hydrolyzed protein, 3.0 g of yeast extract, 1.0 g ofglucose, and 2% agar. The pH was not adjusted; pH before autoclaving wasapproximately 6.8; after autoclaving approximately pH 7.7.

The sucrose/soy seed flask medium (S/S-1) was composed per liter of 65 gof sucrose, 35 g of soy flour, 2 g of Na₃-citrate.2H₂O, 4 g of KH₂PO₄, 5g of Na₂HPO₄, and 6 ml of trace elements. The medium was adjusted pH toabout 7 with NaOH; after dispensing the medium to flasks, 0.2% vegetableoil was added to suppress foaming. Trace elements was composed per literof 100 g of citric acid-H₂O, 20 g of FeSO₄.7H₂O, 5 g of MnSO₄H₂O, 2 g ofCuSO₄.5H₂O, and 2 g of ZnCl₂.

The pH was adjusted to 6.8-7.0 with ammonia before inoculation, andcontrolled thereafter at pH 7.0±0.2 with ammonia and H₃PO₄. Thetemperature was maintained at 37° C. Agitation was at a maximum of 1300RPM using two 6-bladed rushton impellers of 6 cm diameter in 3 litertank with initial volume of 1.5 liters. The aeration had a maximum of1.5 VVM.

For feed, a simple sucrose solution was used. Feed started at about 4hours after inoculation, when dissolved oxygen (D.O.) was still beingdriven down (i.e., before sucrose depletion). The feed rate was rampedlinearly from 0 to approximately 6 g sucrose/L₀-hr over a 7 hour timespan. A lower feed rate, ramped linearly from 0 to approximately 2 gsucrose/L₀-hr, was also used in some fermentations.

Viscosity was noticeable by about 10 hours and by 24 hours viscosity wasvery high, causing the D.O. to bottom-out. End-point viscosity reached3,220 cP. Cell mass development reached a near maximum (12 to 15g/liter) by 20 hours. Cells were removed by diluting 1 part culture with3 parts water, mixing well and centrifuging at about 30,000×g to producea clear supernatant and cell pellet, which can be washed and dried.

Assays of hyaluronic acid concentration were performed using the ELISAmethod, based on a hyaluronan binding protein (protein and kitscommercially available from Seikagaku America, Falmouth, Mass.).

Bacillus subtilis RB 161 and RB163 were cultured in batch and fed-batchfermentations. In the fed-batch processes, the feed rate was variedbetween cultures of Bacillus subtilis strains RB163 and RB161. Assays ofhyaluronic acid concentrations were again performed using the ELISAmethod. The results are provided in Table 2.

TABLE 2 Strain and Growth HA (relative yield) Conditions ELISA methodRB-161 0.7 ± 0.1 (hasA/tuaD/gtaB) simple batch RB-163 0.9 ± 0.1(hasA/tuaD/gcaD) fed batch ~6 g sucrose/L₀-hr RB161 0.9 ± 0.1(hasA/tuaD/gtaB) fed batch ~6 g sucrose/L₀-hr RB-163  1.0 ± 0..2(hasA/tuaD/gcaD) fed batch ~2 g sucrose/L₀-hr RB161  1.0 ± 0..1(hasA/tuaD/gtaB) fed batch ~2 g sucrose/L₀-hr

The results of the culture assays for the same strain at a fed batchrate of 2 g/L sucrose/L₀-hr compared to 6 g/L sucrose/L₀-hr demonstratedthat a faster sucrose feed rate did not significantly improve titers.

A summary of the Bacillus strains run under same conditions (fed batchat approximately 2 g sucrose/L₀-hr, 37° C.) is shown in FIG. 44. In FIG.44, ±values indicate standard deviation of data from multiple runs underthe same conditions. Data without ±values are from single runs.Hyaluronic acid concentrations were determined using the modifiedcarbazole method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334).

A summary of peak hyaluronic acid weight average molecular weights (MDa)obtained from fermentation of the recombinant Bacillus subtilis strainsunder the same conditions (fed batch at approximately 2 g sucrose/L₀-hr,37° C.) is shown in FIG. 45. Molecular weights were determined using aGPC MALLS assay. Data was gathered from GPC MALLS assays using thefollowing procedure. GPC-MALLS (gel permeation or size-exclusion)chromatography coupled with multi-angle laser light scattering) iswidely used to characterize high molecular weight (MW) polymers.Separation of polymers is achieved by GPC, based on the differentialpartitioning of molecules of different MW between eluent and resin. Theaverage molecular weight of an individual polymer is determined by MALLSbased the differential scattering extent/angle of molecules of differentMW. Principles of GPC-MALLS and protocols suited for hyaluronic acid aredescribed by Ueno et al., 1988, Chem. Pharm. Bull. 36, 4971-4975; Wyatt,1993, Anal. Chim. Acta 272: 1-40; and Wyatt Technologies, 1999, “LightScattering University DAWN Course Manual” and “DAWN EOS Manual” WyattTechnology Corporation, Santa Barbara, Calif.). An Agilent 1100isocratic HPLC, a Tosoh Biosep G6000 PWxl column for the GPC, and aWyatt Down EOS for the MALLS were used. An Agilent G1362A refractiveindex detector was linked downstream from the MALLS for eluateconcentration determination. Various commercial hyaluronic acid productswith known molecular weights served as standards.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:

Deposit Accession Number Date of Deposit E. coli XL10 Gold kan (pMRT106)NRRL B-30536 Dec. 12, 2001

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method for producing a hyaluronic acid, comprising: (a) cultivatinga Bacillus host cell under conditions suitable for production of thehyaluronic acid, wherein the Bacillus host cell comprises an artificialoperon comprising a short “consensus” amyQ promoter having the sequenceTTGACA for the “−35” region and TATAAT for the “−10” region operablylinked to a hyaluronan synthase encoding sequence, a UDP-glucose6-dehydrogenase encoding sequence, and a UDP-glucose pyrophosphorylaseencoding sequence; wherein the hyaluronan synthase encoding sequence is(i) a nucleic acid sequence encoding a polypeptide comprising SEQ ID NO:95; or (ii) a nucleic acid sequence which hybridizes under highstringency conditions with SEQ ID NO: 94 or its full-length complement;wherein the UDP-glucose 6-dehydrogenase encoding sequence is (i) anucleic acid sequence encoding a polypeptide comprising SEQ ID NO: 12;or (ii) a nucleic acid sequence which hybridizes under high stringencyconditions with SEQ ID NO: 11 or its full-length complement; wherein theUDP-glucose pyrophosphorylase encoding sequence is (i) a nucleic acidsequence encoding a polypeptide comprising SEQ ID NO: 22; or (ii) anucleic acid sequence which hybridizes under high stringency conditionswith SEQ ID NO: 21 or its full-length complement; and wherein highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmonsperm DNA, and 50% formamide, and washing three times each for 15minutes using 2×SSC, 0.2% SDS preferably at least at 65° C.; and (b)recovering the hyaluronic acid from the cultivation medium.
 2. Themethod of claim 1, wherein the hyaluronan synthase encoding sequenceencodes a polypeptide comprising SEQ ID NO:
 95. 3. The method of claim1, wherein the hyaluronan synthase encoding sequence is a nucleic acidsequence which hybridizes under high or very high stringency conditionswith SEQ ID NO: 94 or its full-length complement; wherein highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmonsperm DNA, and 50% formamide, and washing three times each for 15minutes using 2×SSC, 0.2% SDS preferably at least at 65° C. and whereinvery high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and 50% formamide, and washing three timeseach for 15 minutes using 2×SSC, 0.2% SDS preferably at least at 70° C.4. The method of claim 1, wherein the UDP-glucose 6-dehydrogenaseencoding sequence encodes a polypeptide comprising SEQ ID NO:
 12. 5. Themethod of claim 1, wherein the UDP-glucose 6-dehydrogenase encodingsequence is a nucleic acid sequence which hybridizes under high or veryhigh stringency conditions with SEQ ID NO: 11 or its full-lengthcomplement; wherein high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, andwashing three times each for 15 minutes using 2×SSC, 0.2% SDS preferablyat least at 65° C. and wherein very high stringency conditions aredefined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3%SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50%formamide, and washing three times each for 15 minutes using 2×SSC, 0.2%SDS preferably at least at 70° C.
 6. The method of claim 1, wherein theUDP-glucose pyrophosphorylase encoding sequence encodes a polypeptidecomprising SEQ ID NO:
 22. 7. The method of claim 1, wherein theUDP-glucose pyrophosphorylase encoding sequence is a nucleic acidsequence which hybridizes under high or very high stringency conditionswith SEQ ID NO: 21 or its full-length complement; wherein highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmonsperm DNA, and 50% formamide, and washing three times each for 15minutes using 2×SSC, 0.2% SDS preferably at least at 65° C. and whereinvery high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and 50% formamide, and washing three timeseach for 15 minutes using 2×SSC, 0.2% SDS preferably at least at 70° C.8. The method of claim 1, wherein the artificial operon furthercomprises one or more additional genes encoding enzymes in thebiosynthesis of a precursor sugar of the hyaluronic acid or the Bacillushost cell further comprises one or more nucleic acid constructscomprising one or more additional genes encoding enzymes in thebiosynthesis of a precursor sugar of the hyaluronic acid.
 9. The methodof claim 8, wherein the one or more additional genes encoding enzymes inthe biosynthesis of a precursor sugar of the hyaluronic acid areselected from the group consisting of a UDP-N-acetylglucosaminepyrophosphorylase gene, glucose-6-phosphate isomerase gene, hexokinasegene, phosphoglucomutase gene, amidotransferase gene, mutase gene, andacetyl transferase gene.
 10. The method of claim 9, wherein theUDP-N-acetylglucosamine pyrophosphorylase encoding sequence encodes apolypeptide comprising SEQ ID NO:
 30. 11. The method of claim 9, whereinthe UDP-N-acetylglucosamine pyrophosphorylase encoding sequence is (a) anucleic acid sequence encoding a polypeptide comprising SEQ ID NO: 30;or (b) a nucleic acid sequence which hybridizes under high or very highstringency conditions with SEQ ID NO: 29 or its full-length complement;wherein high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and 50% formamide, and washing three timeseach for 15 minutes using 2×SSC, 0.2% SDS preferably at least at 65° C.and wherein very high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, andwashing three times each for 15 minutes using 2×SSC, 0.2% SDS preferablyat least at 70° C.
 12. The method of claim 9, wherein theglucose-6-phosphate isomerase encoding sequence encodes a polypeptidecomprising SEQ ID NO:
 101. 13. The method of claim 9, wherein theglucose-6-phosphate isomerase encoding sequence is (a) a nucleic acidsequence encoding a polypeptide comprising SEQ ID NO: 101; or (b) anucleic acid sequence which hybridizes under high or very highstringency conditions with SEQ ID NO: 100 or its full-length complement;wherein high stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and 50% formamide, and washing three timeseach for 15 minutes using 2×SSC, 0.2% SDS preferably at least at 65° C.and wherein very high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, andwashing three times each for 15 minutes using 2×SSC, 0.2% SDS preferablyat least at 70° C.
 14. The method of claim 9, wherein the one or moreadditional genes selected from the group of the UDP-N-acetylglucosaminepyrophosphorylase gene, glucose-6-phosphate isomerase gene, hexokinasegene, phosphoglucomutase gene, amidotransferase gene, mutase gene, andacetyl transferase gene are under the control of the same or a differentpromoter(s) as the hyaluronan synthase encoding sequence.
 15. The methodof claim 1, wherein the artificial operon further comprises an mRNAprocessing/stabilizing sequence located downstream of the short“consensus” amyQ promoter operably linked to the hyaluronan synthaseencoding sequence, the UDP-glucose 6-dehydrogenase encoding sequence,and the UDP-glucose pyrophosphorylase encoding sequence and upstream ofthe hyaluronan synthase encoding sequence, the UDP-glucose6-dehydrogenase encoding sequence, and the UDP-glucose pyrophosphorylaseencoding sequence.
 16. The method of claim 1, wherein the artificialoperon further comprises a selectable marker gene.
 17. The method ofclaim 1, wherein the Bacillus host cell is selected from the groupconsisting of Bacillus agaradherens, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis.
 18. The method of claim 1, wherein the Bacillus host cellis unmarked with a selectable marker.
 19. The method of claim 1, whereinthe artificial operon is integrated into the chromosome of the Bacillushost cell.
 20. The method of claim 1, wherein the Bacillus host cell isa Bacillus licheniformis cell or a Bacillus subtilis cell.